Title: BIOPHYSICAL METHODS
1 2- Analysis of Bio molecules
- UV and Visible Light
- Spectroscopy NMR and ESR
- Circular dichorism
- X ray diffraction
- Mass Spectroscopy
- Surface plasmon Resonance
3ELECTROMAGNETIC SPECTRUM
4ELECTROMAGNETIC SPECTUM
5UV SPECTRUM
6VISIBLE SPECRUM
7INFRARED SPECRTUM
8Quantitative UV
- Quantitative UV/vis is used to determine the
concentration of an analyte usually in an aqueous
solution.
9- In order to be able to do this, the analyte must
absorb in the UV/vis region.
10- 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.
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12UV 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 (jm).
13- 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.
14- High-energy electronic transitions are observed
in the low-wavelength UV/VIS regions. - Moderate-energy vibrational and rotational
transitions are observed in the high-wavelength
IR region.
15- 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
16- 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
17Radiation Sources
- The function of the source is to provide
radiation of sufficient energy to
makemeasurements in the region of spectral
interest.
18- 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
19- 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.
20- The energy of the deuterium source is relatively
lower than the energy of the mercury source.
21- The two sources used in the visible and NIR
regions are tungsten filaments and quartz-halide
lamps.
22- 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 narrow-band filters
are used to isolate the wavelengths of interest.
23- 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.
24- 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
25- 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
26The Mono chromator
- Dispersive and nondispersive mono chromator are
used in photometric analysis
27- 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.
28MONO CROMATOR
29SPCTROPHOTOMETER
30- Spectrophotometers are dispersive instruments and
photometers are non-dispersive instruments. - 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
31- 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.
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33- Photometers are non-dispersive devices which
exclude a large amount of spectral radiation. - Photometers are used to make measurements at
selected discrete wavelengths.
34- The measurement wavelength filter is selected to
match the absorption band of the component being
analyzed.
35- 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.
36The Sample Cell
- The purpose of the sample cell is to contain a
representative sample from the process stream.
37- 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.
38Detectors
- Several types of detectors are used in process UV
analyzers, including phototubes, photomultiplier
tubes, and photocells.
39- 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
40- 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
41- 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.
42PHOTOMULTIPLIER
43- 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.
44PHOTODIODE ARRAY
45Readouts
- Analog meters, digital meters, strip chart
recorders, and video display tubes (VDTs) are
examples of readout devices used in photometers
and spectrophotometers
46ANALOGUE METER
47DIGITAL METER
48STRIP CHART RECORDER
49VIDEO DISPLAY TUBE
50Scanning 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
51DIFFRACTION GRATING
52DIFFRACTION GRATING
53SCANNING SPECTROPHOTOMETER
54Spectrometer
- 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
55SPECTROMETER
56UV / VISIBLE SPECTROMETER
57-
- 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.
58Circular 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
59CIRCULAR DICHORISM
60CIRCULAR DICHORISM
61- 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.
62Interaction 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
63- 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.
64Application 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
65- 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
66- 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
67- 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 metalprotein interactions
68- 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
69- CD can be used to survey a large number of
solvent conditions, varying temperature, pH,
salinity, and the presence of various cofactors.
70Nuclear 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
71NMR OVER VIEW
72MAGNETIC RESONANCE
73- 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).
74- All nuclei that contain odd numbers of nucleons
have an intrinsic magnetic moment and angular
momentum, in other words a spin gt 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
75- 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
76- 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
77-
- The required perturbing frequency is dependent
upon the static magnetic field (H0) and the
nuclei of observation.
78- 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
79- 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.
80NMR 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
81- 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
82NMR SPECTROPHOTOMETER
83High magnetic field (800 MHz, 18.8 T) NMR
spectrometer being loaded with a sample.
84- 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
85- 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.
86The NMR sample is prepared in a thin-walled glass
tube - an NMR tube.
87- 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
88- 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.
89- 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.
90Chemical shift
- Depending on the local chemical environment,
different protons in a molecule resonate at
slightly different frequencies
91- 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
92- By understanding different chemical environments,
the chemical shift can be used to obtain some
structural information about the molecule in a
sample
93Correlation 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
94CORRELATION SPECTROSCOPY
95- 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
96Solid-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
97- 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.
98Electron 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
99-
- 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
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101- 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.
102EPR spectrometer
103- 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
104EPR 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
105- 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
106- 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.
107- 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
108- 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.
109X-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.
110X RAY SCATTERING
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112- 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
113- 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.
114X-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
115X RAY DIFFRACTION
116- 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.
117- 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
118- 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
119- 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
120Compton 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
121COMPTON SCATTERING
122- The Compton effect was observed by Arthur Holly
Compton in 1923 - Arthur Compton earned the 1927 Nobel Prize in
Physics for the discovery.
123X-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
124Mass 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
125MASS SPECTROMETRY
126MASS SPECTROMETER
127- The MS principle consists of ionizing chemical
compounds to generate charged molecules or
molecule fragments and measurement of their
mass-to-charge ratios
128Typical 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),
129- 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
130- 5) detection of the ions, which in step 4) were
sorted according to m/z.
131- 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
132- A detector, which measures the value of an
indicator quantity and thus provides data for
calculating the abundances of each ion present
133- 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
134Main steps of measuring with a mass spectrometer
135- 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.
136Tandem mass spectrometry
- A tandem mass spectrometer is one capable of
multiple rounds of mass spectrometry, usually
separated by some form of molecule fragmentation
137TANDEM MASS SPECTROMETER
138- 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
139- 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).
140- An important application using tandem mass
spectrometry is in protein identification
141- 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 spectrometer. - One of the most important applications of this
technique is radiocarbon dating.
142Mass 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
143- Usually the first strategy for identifying an
unknown compound is to compare its experimental
mass spectrum against a library of mass spectra
144- Computer simulation of ionization and
fragmentation processes occurring in mass
spectrometer is the primary tool for assigning
structure or peptide sequence to a molecule
145- 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.
146Applications
- Isotope dating and tracking
- Mass spectrometer to determine the 16O/18O and
12C/13C isotope ratio on biogenous carbonate
147- 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
148- 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).
149- 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
150CHANDRAYAN
151High Resolution Mass Spectrometer
152- Respired gas monitor
- Mass spectrometers were used in hospitals for
respiratory gas analysis beginning around 1975
through the end of the century
153Surface 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.
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157- 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.
158- 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.
159- 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
160- 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
161SPR 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
162Applications
- Surface plasmons have been used to enhance the
surface sensitivity of several spectroscopic
measurements including fluorescence, Raman
scattering, and second harmonic generation
163- in their simplest form, SPR reflectivity
measurements can be used to detect molecular
adsorption, such as polymers, DNA or proteins,
etc
164Magnetic 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.