BIOPHYSICAL METHODS

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 high-wavelength 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 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 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 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 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 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 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 three-dimensional 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 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

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-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 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 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 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 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 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.