BIOPHYSICAL
METHODS
•Analysis of Bio molecules
•UV and Visible Light
• Spectroscopy NMR and ESR
• Circular dichorism
• X ray diffraction
• Mass Spectroscopy
• Surface plasmon Resonance
ELECTROMAGNETIC
SPECTRUM
ELECTROMAGNETIC
SPECTUM
UV SPECTRUM
VISIBLE SPECRUM
INFRARED SPECRTUM
Quantitative UV
• Quantitative UV/vis is used
to determine the
concentration of an analyte
usually in an aqueous
solution.
• 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
concentration.
• 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
• UV ANALYZERS
• 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
transitions.
• High-energy electronic
transitions are observed in the
low-wavelength UV/VIS
regions.
• Moderate-energy vibrational
and rotational transitions are
observed in the highwavelength IR region.
• The Main Components of UV
Analyzers
• 1. Source-provides radiation for
the spectral region being
measured
• 2. Mono chromator-a device
used to select narrow bands of
wavelengths
• 3. Sample cell-contains the
sample at an appropriate path
length
• 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
interest.
• 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
region.
•The energy of the deuterium
source is relatively lower than
the energy of the mercury
source.
•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
sources.
• The broad emission source
provides energy in a broad
wavelength band, and narrowband 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
wavelength.
• 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
agent.
MONO CROMATOR
SPCTROPHOTOMETER
•Spectrophotometers are
dispersive instruments and
photometers are non-dispersive
instruments.
•The function of the monochromator 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
wavelengths.
• They can be used to make
measurements at several
wavelengths
• 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
photometer.
• 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.
•
Detectors
• Several types of detectors are
used in process UV analyzers,
including phototubes,
photomultiplier tubes, and
photocells.
• The photoelectric effect is used
in the vacuum phototube to
produce a current proportional to
the energy striking the tube
cathode
• 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
instruments.
• The photoelectric effect is used in
the PMT to produce a current
proportional to the radiation
striking the cathode of the tube.
PHOTOMULTIPLIER
• A recent development in photometric
analyzers is the use of photodiode arrays
(PDA).
• 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
simultaneously.
PHOTODIODE ARRAY
Readouts
• Analog meters, digital meters,
strip chart recorders, and video
display tubes (VDTs) are
examples of readout devices
used in photometers and
spectrophotometers
ANALOGUE METER
DIGITAL METER
STRIP CHART RECORDER
VIDEO DISPLAY TUBE
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
regions
DIFFRACTION GRATING
DIFFRACTION GRATING
SCANNING
SPECTROPHOTOMETER
Spectrometer
• 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
materials
SPECTROMETER
UV / VISIBLE SPECTROMETER
• 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
infrared
• 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
light.
• A CD Spectrometer is an
instrument that records this
phenomenon as a function of
wavelength
CIRCULAR DICHORISM
CIRCULAR DICHORISM
•
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
molecules
• 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
components
• 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
structures
• 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
conformation
• 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
spectroscopy
• 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
cofactors.
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
NMR OVER VIEW
MAGNETIC RESONANCE
• 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
field
• If a sample is placed in a nonuniform 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
H0.
• 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 threedimensional structure of
molecules in solution and the
solid state
• Thus, structural and dynamic
information is obtainable
NMR SPECTROPHOTOMETER
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
mechanisms
• 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
state.
The NMR sample is prepared in a
thin-walled glass tube - an NMR
tube.
• 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, COSY
CORRELATION
SPECTROSCOPY
• 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
resonance
• 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
resonance
• 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
ion
• 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
atomic
• 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
University.
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
centers
• EPR is a sensitive, specific method
for studying both radicals formed in
chemical reactions and the reactions
themselves
• 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
teeth.
• Radiation damage over long
periods of time creates free
radicals in tooth enamel, which
can then be examined by EPR
and, after proper calibration,
dated
• 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
• 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
X RAY DIFFRACTION
• 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
proteins.
• 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
Data
• Thin film diffraction and grazing
incidence X-ray diffraction may be
used to characterize the
crystallographic structure and
preferred orientation of substrateanchored 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
COMPTON SCATTERING
• The Compton effect was
observed by Arthur Holly
Compton in 1923
• Arthur Compton earned the 1927
Nobel Prize in Physics for the
discovery.
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
electrons
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
compounds
MASS SPECTROMETRY
MASS SPECTROMETER
• The MS principle consists of
ionizing chemical compounds to
generate charged molecules or
molecule fragments and
measurement of their mass-tocharge 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-tocharge 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
m/z.
• 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
present
• 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
compounds.
Tandem mass spectrometry
• A tandem mass spectrometer is
one capable of multiple rounds of
mass spectrometry, usually
separated by some form of
molecule fragmentation
TANDEM MASS SPECTROMETER
• 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 collisioninduced 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 megavolt range, to accelerate negative ions
into a type of tandem mass
spectrometer.
• 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
techniques
• 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.
Applications
• Isotope dating and tracking
• Mass spectrometer to determine
the 16O/18O and 12C/13C
isotope ratio on biogenous
carbonate
• 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
CHANDRAYAN
High Resolution Mass
Spectrometer
• 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 nanometersized 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
surface.
• In order to excite surface
plasmons in a resonant manner,
one can use an electron or light
beam (visible and infrared are
typical
• 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
light
• This emitted light can be detected
behind the metal film in various
directions
Applications
• Surface plasmons have been
used to enhance the surface
sensitivity of several
spectroscopic measurements
including fluorescence, Raman
scattering, and second harmonic
generation
•
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.