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biophysical-methods-2 by pengxuebo

VIEWS: 10 PAGES: 164

•Analysis of Bio molecules
•UV and Visible Light
• Spectroscopy NMR and ESR
• Circular dichorism
• X ray diffraction
• Mass Spectroscopy
• Surface plasmon Resonance
       Quantitative UV

• Quantitative UV/vis is used
  to determine the
  concentration of an analyte
  usually in an aqueous
• In order to be able to do this,
  the analyte must absorb in
  the UV/vis region.
• Beer's Law is a linear relationship
  between absorbance and
• A = a * b * c, where c is
  concentration, A is absorbance, b
  is path length (usually 1 cm) and
  a is the molar absorbtivity.
• Beer's law is linear.
UV Analysis - General Theory
• The UV region consists of
  wavelengths from 200 to 400
  nanometers (nm).
• The visible region extends from
  400 to 800 nm, and the near IR
  (NIR) region covers 0.8 to 2.50
  micrometers (j~m).
• The UVVIS-NIR is a relatively
  small part of the electromagnetic
  radiation spectrum, and the
  shorter the wavelength the more
  penetrating the radiation.
• The region where a compound
  absorbs radiation depends on the
  energy of the molecular
• High-energy electronic
  transitions are observed in the
  low-wavelength UV/VIS
• Moderate-energy vibrational
  and rotational transitions are
  observed in the high-
  wavelength IR region.
• The Main Components of UV
• 1. Source-provides radiation for
  the spectral region being
• 2. Mono chromator-a device
  used to select narrow bands of
• 3. Sample cell-contains the
  sample at an appropriate path
• 4. Detector-a device which
  measures transmitted energy and
  converts it into electrical energy
• 5. Readout device-provides a
  means of recording the
  measurement results
        Radiation Sources
• The function of the source is to
  provide radiation of sufficient
  energy to makemeasurements
  in the region of spectral
• The cadmium, mercury, and
  zinc vapor sources that are
  used in the UV region are
  emission line sources.
• The output of these sources
  provides radiation as narrow
  discrete emission lines at a
  high-energy level
•  Mercury vapor lamps are often
  used because of their long
  service life.
• Deuterium arc sources provide
  a broad band of UV radiation at
  all of the wavelengths in the UV
•The energy of the deuterium
 source is relatively lower than
 the energy of the mercury
•The two sources used in the
 visible and NIR regions are
 tungsten filaments and
 quartz-halide lamps.
• Two types of UV energy sources
  are used:
•broad and discrete line emission
• The broad emission source
  provides energy in a broad
  wavelength band, and narrow-
  band filters are used to isolate the
  wavelengths of interest.
• These sources provide all
  wavelengths in the region but
  usually have a low-emission, or
  low-energy level, at any given
• Sources of this type include
  hydrogen, or deuterium,
  discharge lamps; tungsten lamps;
  and tungsten-iodine lamps.
• Discrete line sources use gas
  discharge lamps with narrow lines
  of emission.
• These sources emit radiation energy
  at various discrete wavelengths at a
  high-energy level
• The wavelengths that are not
  desired are filtered, leaving only
  the wavelength of interest
• Tungsten-iodine cycle lamps can
  be used down to 300 nm
• Mercury vapor lamps are the
  most useful UV sources due to
  their high intensity and long life
• Medium-pressure mercury lamps
  can operate down to 300 nm
• Zinc discharge lamps are useful
  due to their 214 nm emission line
    The Mono chromator

•Dispersive and nondispersive
 mono chromator are used in
 photometric analysis
• A monochromator is an optical device
  that transmits a mechanically
  selectable narrow band of
  wavelengths of light or other radiation
  chosen from a wider range of
  wavelengths available at the input.
  The name is from the Greek roots
  mono-, single, and chroma, colour,
  and the Latin suffix -ator, denoting an
•Spectrophotometers are
 dispersive instruments and
 photometers are non-dispersive
•The function of the mono-
 chromator is to disperse light
 from a source and selectively pass
 a narrow spectral band to the
 sample and detector
• Spectrophotometers are
  dispersive devices that are used
  to scan across a spectrum of
• They can be used to make
  measurements at several
• This capability allows for the
  analysis of multiple components
  with a spectrophotometer.
• Photometers are non-dispersive
  devices which exclude a large
  amount of spectral radiation.
• Photometers are used to make
  measurements at selected
  discrete wavelengths.
•    The measurement wavelength
    filter is selected to match the
    absorption band of the
    component being analyzed.
• The ratio of the transmitted light
  at the reference and measured
  wavelengths is measured by the
• Normally, photometers are used
  to measure a single component in
  a process stream.
        The Sample Cell
•The purpose of the sample cell is
 to contain a representative
 sample from the process stream.
• Stainless steel is the material most
  commonly used for cell bodies
• Other metals such as Monel,
  Hastelloy, and titanium are also used.
• Plastic cell bodies made of Teflon or
  Kynar are used in some applications
• Quartz, sapphire, and glass cell
  windows are used in the UV-VIS-NIR
  spectral regions.
• Several types of detectors are
  used in process UV analyzers,
  including phototubes,
  photomultiplier tubes, and
• The photoelectric effect is used
  in the vacuum phototube to
  produce a current proportional to
  the energy striking the tube
• The photomultiplier tube offers
  very sensitive detection of UV and
  visible light but large radiation
  energy levels will damage the
  light-sensitive surface
• The photocell (photovoltaic) is a
  semiconductor light detector of
  the barrier layer type
• A current is developed
  proportional to the light intensity
  but, the current output is not
  linear with the energy level
• Photomultiplier tubes (PMT) have
  traditionally been used in UV/VIS
• The photoelectric effect is used in
  the PMT to produce a current
  proportional to the radiation
  striking the cathode of the tube.
• A recent development in photometric
  analyzers is the use of photodiode arrays
• The PDA detectors are used throughout
  UV-VIS-NIR regions.
• A large number of discrete detectors are
  located in a very close space in the PDA
• This array of diode detectors allows for all
  of the wavelengths to be measured
• Analog meters, digital meters,
  strip chart recorders, and video
  display tubes (VDTs) are
  examples of readout devices
  used in photometers and
 Scanning Spectrophotometers
• Scanning spectrophotometers are
  dispersive devices that normally
  utilize diffraction gratings to scan
  across a spectral region
• Scanning devices can be used for
  multiple component applications
• . Scanning spectrophotometers can
  be used in the UV, visible, and NIR
• A spectrograph is an optical
  instrument used to measure
  properties of light over a specific
  portion of the electromagnetic
  spectrum, typically used in
  spectroscopic analysis to identify
• Spectrometer is a term that is applied
  to instruments that operate over a
  very wide range of wavelengths, from
  gamma rays and X-rays into the far
• If the region of interest is restricted to
  near the visible spectrum, the study is
  called spectrophotometry.
       Circular dichroism
• Circular dichroism (CD) is the
  differential absorption of left- and
  right-handed circularly polarized
• A CD Spectrometer is an
  instrument that records this
  phenomenon as a function of
• CD can be used to help
  determine the structure of
  macromolecules (including the
  secondary structure of proteins
  and the handedness of DNA).
• CD was discovered by the French
  physicist Aimé Cotton in 1896.
Interaction of circularly polarized
         light with matter
• The electric field of a light beam
  causes a linear displacement of
  charge when interacting with a
  molecule, whereas the magnetic field
  of it causes a circulation of charge
• These two motions combined result in
  a helical displacement when light
  impinges on a molecule
• The two types of circularly polarized light
  are absorbed to different extents
• In a CD experiment, equal amounts of left
  and right circularly polarized light of a
  selected wavelength are alternately
  radiated into a (chiral) sample
• One of the two polarizations is absorbed
  more than the other one, and this
  wavelength-dependent difference of
  absorption is measured, yielding the CD
  spectrum of the sample.
      Application to biological
• In general, this phenomenon will
  be exhibited in absorption bands
  of any optically active molecule.
• As a consequence, circular
  dichroism is exhibited by
  biological molecules, because of
  their dextrorotary and levorotary
• Even more important is that a secondary
  structure will also impart a distinct CD to
  its respective molecules.
• Therefore, the alpha helix of proteins and
  the double helix of nucleic acids have CD
  spectral signatures representative of their
• The far-UV (ultraviolet) CD spectrum of
  proteins can reveal important
  characteristics of their secondary structure
• CD spectra can be readily used to
  estimate the fraction of a
  molecule that is in the alpha-helix
  conformation, the beta-sheet
  conformation, the beta-turn
  conformation, or some other (e.g.
  random coil) conformation
• It can reveal important
  thermodynamic information
• CD a valuable tool for verifying
  that the protein is in its native
• Visible CD spectroscopy is a very
  powerful technique to study
  metal–protein interactions
• CD gives less specific structural
  information than X-ray
  crystallography and protein NMR
• for example, which both give
  atomic resolution data
• However, CD spectroscopy is a
  quick method that does not
  require large amounts of
•   CD can be used to survey a large
    number of solvent conditions,
    varying temperature, pH, salinity,
    and the presence of various
 Nuclear magnetic resonance
• Nuclear magnetic resonance (NMR)
  is the name given to a physical
  resonance phenomenon involving the
  observation of specific quantum
  mechanical magnetic properties of an
  atomic nucleus in the presence of an
  applied, external magnetic field
• Many scientific techniques exploit
  NMR phenomena to study
  molecular physics, crystals and
  non-crystalline materials through
  NMR spectroscopy
• NMR is also routinely used in
  advanced medical imaging
  techniques, such as in magnetic
  resonance imaging (MRI).
• All nuclei that contain odd numbers of
  nucleons have an intrinsic magnetic
  moment and angular momentum, in
  other words a spin > 0.
• The most commonly studied nuclei
  are 1H
• A key feature of NMR is that the
  resonance frequency of a particular
  substance is directly proportional to
  the strength of the applied magnetic
• If a sample is placed in a non-
  uniform magnetic field then the
  resonance frequencies of the
  sample's nuclei depend on where
  in the field they are located

• The principle of NMR usually
  involves two sequential steps:
• The alignment (polarization) of
  the magnetic nuclear spins in an
  applied, constant magnetic field
• The perturbation of this alignment
  of the nuclear spins by employing
  an electro-magnetic, usually radio
  frequency (RF) pulse
• The required perturbing frequency
  is dependent upon the static
  magnetic field (H0) and the nuclei
  of observation.
• The two fields are usually chosen to
  be perpendicular to each other as this
  maximises the NMR signal strength
• The resulting response by the total
  magnetization (M) of the nuclear
  spins is the phenomenon that is
  exploited in NMR spectroscopy and
  magnetic resonance imaging
• NMR phenomena are also utilized
  in low-field NMR, NMR
  spectroscopy and MRI in the
  Earth's magnetic field (referred to
  as Earth's field NMR), and in
  several types of magnetometers.
        NMR spectroscopy
• NMR spectroscopy is one of the principal
  techniques used to obtain physical,
  chemical, electronic and structural
  information about molecules due to either
  the chemical shift Zeeman effect, or the
  Knight shift effect, or a combination of
  both, on the resonant frequencies of the
  nuclei present in the sample
•  It is a powerful technique that can
  provide detailed information on
  the topology, dynamics and three-
  dimensional structure of
  molecules in solution and the
  solid state
• Thus, structural and dynamic
  information is obtainable
High magnetic field (800 MHz, 18.8 T) NMR
 spectrometer being loaded with a sample.
• Nuclear magnetic resonance
  spectroscopy, most commonly
  known as NMR spectroscopy, is
  the name given to a technique
  which exploits the magnetic
  properties of certain nuclei
• Many types of information can be
  obtained from an NMR spectrum
• It can, among other things, be used
  to study mixtures of analytes, to
  understand dynamic effects such as
  change in temperature and reaction
• It is an invaluable tool in
  understanding protein and nucleic
  acid structure and function. It can be
  applied to a wide variety of samples,
  both in the solution and the solid
The NMR sample is prepared in a
 thin-walled glass tube - an NMR
• When placed in a magnetic field,
  NMR active nuclei (such as 1H or
  13C) absorb at a frequency
  characteristic of the isotope.
• The resonant frequency, energy
  of the absorption and the intensity
  of the signal are proportional to
  the strength of the magnetic field
• For example, in a 21 tesla
  magnetic field, protons resonate
  at 900 MHz. It is common to refer
  to a 21 T magnet as a 900 MHz
  magnet, although different nuclei
  resonate at a different frequency
  at this field strength.
• In the Earth's magnetic field the
  same nuclei resonate at audio
  frequencies. This effect is used in
  Earth's field NMR spectrometers
  and other instruments.
         Chemical shift
• Depending on the local chemical
  environment, different protons in
  a molecule resonate at slightly
  different frequencies
• Since both this frequency shift
  and the fundamental resonant
  frequency are directly proportional
  to the strength of the magnetic
  field, the shift is converted into a
  field-independent dimensionless
  value known as the chemical shift
• By understanding different
  chemical environments, the
  chemical shift can be used to
  obtain some structural information
  about the molecule in a sample
  Correlation spectroscopy
• Correlation spectroscopy is one of
  several types of two-dimensional
  nuclear magnetic resonance
  (NMR) spectroscopy
• This type of NMR experiment is
  best known by its acronym,
• Other types of two-dimensional NMR
  include J-spectroscopy, exchange
  spectroscopy (EXSY), Nuclear
  Overhauser effect spectroscopy
  (NOESY), total correlation
  spectroscopy (TOCSY) and
  heteronuclear correlation
  experiments, such as HSQC, HMQC,
  and HMBC
  Solid-state nuclear magnetic
• A variety of physical
  circumstances does not allow
  molecules to be studied in
  solution, and at the same time not
  by other spectroscopic techniques
  to an atomic level
• Applications in which solid-state NMR
  effects occur are often related to
  structure investigations on membrane
  proteins, protein fibrils or all kinds of
  polymers, and chemical analysis in
  inorganic chemistry, but also include
  "exotic" applications like the plant
  leaves and fuel cells.
       Electron paramagnetic
• Electron paramagnetic resonance (EPR)
  or electron spin resonance (ESR)
  spectroscopy is a technique for studying
  chemical species that have one or more
  unpaired electrons, such as organic and
  inorganic free radicals or inorganic
  complexes possessing a transition metal
• The basic physical concepts of
  EPR are analogous to those of
  nuclear magnetic resonance
  (NMR), but it is electron spins that
  are excited instead of spins of
• EPR was first observed in Kazan
  State University by a Soviet
  physicist Yevgeny Zavoisky in
  1944, It was developed
  independently at the same time
  by Brebis Bleaney at Oxford
EPR spectrometer
• In principle, EPR spectra can be
  generated by either varying the
  photon frequency incident on a
  sample while holding the
  magnetic field constant, or doing
  the reverse
        EPR applications
• EPR spectroscopy is used in
  various branches of science, such
  as chemistry and physics, for the
  detection and identification of free
  radicals and paramagnetic
• EPR is a sensitive, specific method
  for studying both radicals formed in
  chemical reactions and the reactions
• For example, when frozen water
  (solid H2O) is decomposed by
  exposure to high-energy radiation,
  radicals such as H, OH, and HO2 are
  produced. Such radicals can be
  identified and studied by EPR
• Organic and inorganic radicals can be
  detected in electrochemical systems and
  in materials exposed to UV light
• Medical and biological applications of EPR
  also exist
• Specially-designed nonreactive radical
  molecules can attach to specific sites in a
  biological cell, and EPR spectra can then
  give information on the environment of
  these so-called spin-label or spin-probes.
• EPR also has been used by
  archaeologists for the dating of
• Radiation damage over long
  periods of time creates free
  radicals in tooth enamel, which
  can then be examined by EPR
  and, after proper calibration,
• Radiation-sterilized foods have
  been examined with EPR
  spectroscopy, the aim being to
  develop methods to determine if a
  particular food sample has been
  irradiated and to what dose.
    X-ray scattering techniques
• This is an X-ray diffraction pattern
  formed when X-rays are focused
  on a crystalline material, in this
  case a protein
• Each dot, called a reflection,
  forms from the coherent
  interference of scattered X-rays
  passing through the crystal.
• X-ray scattering techniques are a
  family of non-destructive
  analytical techniques which reveal
  information about the
  crystallographic structure,
  chemical composition, and
  physical properties of materials
  and thin films
•   These techniques are based on
    observing the scattered intensity
    of an X-ray beam hitting a sample
    as a function of incident and
    scattered angle, polarization, and
    wavelength or energy.
 X-ray diffraction techniques
• X-ray diffraction finds the
  geometry or shape of a molecule
  using X-rays.
• X-ray diffraction techniques are
  based on the elastic scattering of
  X-rays from structures that have
  long range order
• Single-crystal X-ray diffraction is a
  technique used to solve the
  complete structure of crystalline
  materials, ranging from simple
  inorganic solids to complex
  macromolecules, such as
• Powder diffraction (XRD) is a
  technique used to characterize the
  crystallographic structure, crystallite
  size (grain size)
• Powder diffraction is commonly used
  to identify unknown substances, by
  comparing diffraction data against a
  database maintained by the
  International Centre for Diffraction
• Thin film diffraction and grazing
  incidence X-ray diffraction may be
  used to characterize the
  crystallographic structure and
  preferred orientation of substrate-
  anchored thin films
• High-resolution X-ray diffraction is
  used to characterize thickness,
  crystallographic structure, and strain
  in thin epitaxial films. It employs
  parallel-beam optics
• X-ray pole figure analysis enables
  one to analyze and determine the
  distribution of crystalline orientations
  within a crystalline thin-film sample
      Compton scattering
• Compton scattering or the Compton
  effect is the decrease in energy
  (increase in wavelength) of an X-ray
  or gamma ray photon, when it
  interacts with matter
• Compton scattering usually refers to
  the interaction involving only the
  electrons of an atom
• The Compton effect was
  observed by Arthur Holly
  Compton in 1923
• Arthur Compton earned the 1927
  Nobel Prize in Physics for the
     X-ray Raman scattering
• X-ray Raman scattering (XRS) is non-resonant
  inelastic scattering of x-rays from core electrons
• .
• It is analogous to Raman scattering, which is a
  largely-used tool in optical spectroscopy, with
  the difference being that the wavelengths of the
  exciting photons fall in the x-ray regime and the
  corresponding excitations are from deep core
    Mass spectrometry (MS)
• Mass spectrometry (MS) is an
  analytical technique for the
  determination of the elemental
  composition of a sample or molecule
• It is also used for elucidating the
  chemical structures of molecules,
  such as peptides and other chemical
• The MS principle consists of
  ionizing chemical compounds to
  generate charged molecules or
  molecule fragments and
  measurement of their mass-to-
  charge ratios
       Typical MS procedure
• 1) a sample is loaded onto the MS
  instrument, and
• 2) the components of the sample
  ionized by one of a variety of methods
  (e.g., by impacting them with an
  electron beam), which results in the
  formation of charged particles (ions),
• 3) directing the ions into a electric
  and/or magnetic fields
• 4) computation of the mass-to-
  charge ratio of the particles based
  on the details of their motion of
  the ions as they transit through
  electromagnetic fields
• 5) detection of the ions, which in
  step 4) were sorted according to
• MS instruments consist of three
  modules: 1.An ion source, which can
  convert gas phase sample molecules
  into ions
• A mass analyzer, which sorts the
  ions by their masses by applying
  electromagnetic fields
• A detector, which measures the
  value of an indicator quantity and
  thus provides data for calculating
  the abundances of each ion
• The technique has both
  qualitative and quantitative uses
• These include identifying
  unknown compounds,
  determining the isotopic
  composition of elements in a
  molecule, and determining the
  structure of a compound by
  observing its fragmentation
Main steps of measuring with a
     mass spectrometer
• Other uses include quantifying the
  amount of a compound in a sample or
  studying the fundamentals of gas
  phase ion chemistry
• MS is now in very common use in
  analytical laboratories that study
  physical, chemical, or biological
  properties of a great variety of
  Tandem mass spectrometry
• A tandem mass spectrometer is
  one capable of multiple rounds of
  mass spectrometry, usually
  separated by some form of
  molecule fragmentation
• For example, one mass analyzer can
  isolate one peptide from many entering a
  mass spectrometer.
• A second mass analyzer then stabilizes
  the peptide ions while they collide with a
  gas, causing them to fragment by collision-
  induced dissociation (CID).
• A third mass analyzer then sorts the
  fragments produced from the peptides
• There are various methods for
  fragmenting molecules for tandem
  MS, including collision-induced
  dissociation (CID), electron capture
  dissociation (ECD), electron transfer
  dissociation (ETD), infrared
  multiphoton dissociation (IRMPD) and
  blackbody infrared radiative
  dissociation (BIRD).
• An important application using
  tandem mass spectrometry is in
  protein identification
• An important type of Tandem mass
  spectrometry is Accelerator Mass
  Spectrometry (AMS), which uses very
  high voltages, usually in the mega-
  volt range, to accelerate negative ions
  into a type of tandem mass
• One of the most important
  applications of this technique is
  radiocarbon dating.
    Mass spectrum analysis
• Since the precise structure or
  peptide sequence of a molecule is
  deciphered through the set of
  fragment masses, the
  interpretation of mass spectra
  requires combined use of various
• Usually the first strategy for
  identifying an unknown compound
  is to compare its experimental
  mass spectrum against a library
  of mass spectra
• Computer simulation of ionization
  and fragmentation processes
  occurring in mass spectrometer is
  the primary tool for assigning
  structure or peptide sequence to
  a molecule
• Another way of interpreting mass
  spectra involves spectra with
  accurate mass
• A computer algorithm called
  formula generator calculates all
  molecular formulas that
  theoretically fit a given mass with
  specified tolerance.
• Isotope dating and tracking
• Mass spectrometer to determine
  the 16O/18O and 12C/13C
  isotope ratio on biogenous
• Pharmacokinetics
• Pharmacokinetics is often studied
  using mass spectrometry
  because of the complex nature of
  the matrix (often blood or urine)
  and the need for high sensitivity
  to observe low dose and long
  time point data
• Protein characterization
• Mass spectrometry is an
  important emerging method for
  the characterization of proteins.
  The two primary methods for
  ionization of whole proteins are
  electrospray ionization (ESI) and
  matrix-assisted laser
  desorption/ionization (MALDI).
• Space exploration
• As a standard method for
  analysis, mass spectrometers
  have reached other planets and
  moons. Two were taken to Mars
  by the Viking program
High Resolution Mass
• Respired gas monitor
• Mass spectrometers were used in
  hospitals for respiratory gas
  analysis beginning around 1975
  through the end of the century
 Surface plasmon resonance
• The excitation of surface
  plasmons by light is denoted as a
  surface plasmon resonance
  (SPR) for planar surfaces or
  localized surface plasmon
  resonance (LSPR) for nanometer-
  sized metallic structures.
• This phenomenon is the basis of
  many standard tools for
  measuring adsorption of material
  onto planar metal (typically gold
  and silver) surfaces or onto the
  surface of metal nanoparticles.
• It is behind many color based
  biosensor applications and
  different lab-on-a-chip sensors.
• Surface plasmons, also known as surface
  plasmon polaritons, are surface
  electromagnetic waves that propagate in a
  direction parallel to the metal/dielectric (or
  metal/vacuum) interface
• Since the wave is on the boundary of the
  metal and the external medium , these
  oscillations are very sensitive to any
  change of this boundary, such as the
  adsorption of molecules to the metal
• In order to excite surface
  plasmons in a resonant manner,
  one can use an electron or light
  beam (visible and infrared are
• The incoming beam has to match
  its impulse to that of the plasmon
• In the case of p-polarized light
  (polarization occurs parallel to the
  plane of incidence), this is
  possible by passing the light
  through a block of glass to
  increase the wavenumber and
  achieve the resonance at a given
  wavelength and angle
           SPR Emission
• When the surface plasmon wave
  hits a local particle or irregularity -
  like on a rough surface-, part of
  the energy can be reemitted as
• This emitted light can be detected
  behind the metal film in various
• Surface plasmons have been
  used to enhance the surface
  sensitivity of several
  spectroscopic measurements
  including fluorescence, Raman
  scattering, and second harmonic
•    in their simplest form, SPR
    reflectivity measurements can be
    used to detect molecular
    adsorption, such as polymers,
    DNA or proteins, etc
 Magnetic Plasmon Resonance
• Recently, there has been an interest
  in magnetic surface plasmons
• These require materials with large
  negative magnetic permeability, a
  property that has only recently been
  made available with the construction
  of metamaterials.

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