BT 11: principle, apparatus, applications and limitations of various biophysical

techniques
Biophysics is an interdisciplinary science using methods of, and theories from, physics to
study biological systems. Biophysics spans all scales of biological organization, from the
molecular scale to whole organisms and ecosystems.
Biophysical research shares significant overlap with biochemistry, nanotechnology,
bioengineering, computational biology and (complex) systems biology.
It has been suggested as a bridge between biology and physics.
Biophysical techniques:
Biophysical methods are techniques to study the structure, properties, dynamics or
function of biomolecules at an atomic or molecular level. They encompass a range of
techniques including microscopy, spectroscopy, electrophysiology, single-molecule
methods and molecular modelling.
There include:
Microscopy, gel electrophoresis, chromatography, cell disruption methods, centrifugation,
NMR, crystallography etc.
Principle, apparatus, applications and limitations of various biophysical techniques
I)
Centrifugation:
Centrifugation is a process which involves the use of the centrifugal force for the
sedimentation of heterogeneous mixtures with a centrifuge, used in industry and in
laboratory settings. This process is used to separate two immiscible liquids. More-dense
components of the mixture migrate away from the axis of the centrifuge, while less-dense
components of the mixture migrate towards the axis. Chemists and biologists may
increase the effective gravitational force on a test tube so as to more rapidly and
completely cause the precipitate (pellet) to gather on the bottom of the tube. The
remaining solution is properly called the "supernate" or "supernatant liquid". The
supernatant liquid is then either quickly decanted from the tube without disturbing the
precipitate, or withdrawn with a Pasteur pipette.
The rate of centrifugation is specified by the angular velocity measured in revolutions per
minute (RPM), or acceleration expressed as g. The conversion factor between RPM and g
depends on the radius of the centrifuge rotor. The particles' settling velocity in
centrifugation is a function of their size and shape, centrifugal acceleration, the volume
fraction of solids present, the density difference between the particle and the liquid, and
the viscosity. The most common application is the separation of solid from highly
concentrated suspensions, which is use in the treatment of sewage sludges for dewatering
where less consistent sediment is produced.
Centrifuges are further classified into various subtypes depending on the speed, rotor
types and intended applications.
Apparatus:

A typical bench top centrifuge separate or concentrate substances suspended in a liquid

medium by density. Space-saving fixed- and variable-speed benchtop or tabletop
centrifuges are used for applications including tissue culture, protein work, DNA/RNA
research, and cell harvesting. Although spinning is used to achieve separation in all
centrifuges, the rotors rpm only indicates the power of the motor. The best indication of
separation power is its RCF, or relative centrifugal force. Versatile multipurpose
centrifuges are the most common type, with an RCF up to about 24,000 g, a variety of
volume ranges, and the ability to spin plates. They can accommodate different types of
rotors, including fixed angle, swinging bucket, and continuous flow. Ultraspeed
centrifuges offer g-forces up to 1,000,000 g, useful in nanotechnology.
Microcentrifuges spin small sample volumes, such as 0.2-mL PCR tubes, at very high
speeds. Other factors to consider include noise level, easy bowl access, refrigeration
capabilities, and rotor material, which can be metal, plastic, or composite.
Applications of centrifugation
Clinical and blood banking
Microbiology
Tissue culture
Molecular biology and genomics
Drug discovery and proteomics

Limitations of centrifugation
Limitations of the centrifuge include high capital and operational costs as they are
energy-intensive. In addition, the maintenance costs and number of incidents of
breakdown are higher than with static separators due to the moving parts.
Another limitation is the narrow range for optimum performance with variable conditions
like feed acceleration, positioning of the interface and solid discharge method. In
addition, sealing materials, especially the dynamic sealing materials, must be carefully
chosen to be chemically and thermally resistant.; therefore, a much more extensive design

and optimization program is required than with the static separators; therefore, a much
more extensive design and optimization program is required than with the static
separators.
The actual achieved separative power of gas centrifuge will be always lower than its
theoretical separative power reflecting additional inefficiencies in the centrifuges when
running as individual machines and in cascades. For example, The IR-1 centrifuge
achieves an average separative power in cascades of less than one SWU/year,
significantly less than its theoretical maximum separative power of 4.9 SWU/year.
Although its value when run individually is greater, it is still far below the theoretical
value. It also experiences a relatively high breakage rate, which accounts for much of the
additional reduction of its separative power when run in production cascades.
II) Gel electrophoresis:
Gel electrophoresis is a method for separation and analysis of macromolecules (DNA,
RNA and proteins) and their fragments, based on their size and charge. It is used in
clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size
independent) and in biochemistry and molecular biology to separate a mixed population
of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments
or to separate proteins by charge.
Nucleic acid molecules are separated by applying an electric field to move the negatively
charged molecules through a matrix of agarose or other substances. Shorter molecules
move faster and migrate farther than longer ones because shorter molecules migrate more
easily through the pores of the gel. This phenomenon is called sieving.
Proteins are separated by charge in agarose because the pores of the gel are too large to
sieve proteins.
Gel electrophoresis can also be used for separation of nanoparticles.
Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium
during electrophoresis, the movement of a charged particle in an electrical field. Gels
suppress the thermal convection caused by application of the electric field, and can also
act as a sieving medium, retarding the passage of molecules; gels can also simply serve to
maintain the finished separation, so that a post electrophoresis stain can be applied.[3]
DNA Gel electrophoresis is usually performed for analytical purposes, often after
amplification of DNA via PCR, but may be used as a preparative technique prior to use of
other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or
Southern blotting for further characterization.
Apparatus:

In simple terms, electrophoresis is a process which enables the sorting of molecules based
on size. Using an electric field, molecules (such as DNA) can be made to move through a
gel made of agar or polyacrylamide. The electric field consists of a negative charge at one
end, which pushes the molecules through the gel, and a positive charge at the other end
that pulls the molecules through the gel. The molecules being sorted are dispensed into a
well in the gel material. The gel is placed in an electrophoresis chamber, which is then
connected to a power source. When the electric current is applied, the larger molecules
move more slowly through the gel while the smaller molecules move faster. The different
sized molecules form distinct bands on the gel.
The term "gel" in this instance refers to the matrix used to contain, then separate the
target molecules. In most cases, the gel is a cross-linked polymer whose composition and
porosity is chosen based on the specific weight and composition of the target to be
analyzed. When separating proteins or small nucleic acids (DNA, RNA, or
oligonucleotides) the gel is usually composed of different concentrations of acrylamide
and a cross-linker, producing different sized mesh networks of polyacrylamide. When
separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is
purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in
contrast to polyacrylamide, is a neurotoxin and must be handled using appropriate safety
precautions to avoid poisoning. Agarose is composed of long unbranched chains of
uncharged carbohydrate without cross links resulting in a gel with large pores allowing
for the separation of macromolecules and macromolecular complexes.
"Electrophoresis" refers to the electromotive force (EMF) that is used to move the
molecules through the gel matrix. By placing the molecules in wells in the gel and
applying an electric field, the molecules will move through the matrix at different rates,
determined largely by their mass when the charge to mass ratio (Z) of all species is
uniform. However, when charges are not all uniform then, the electrical field generated
by the electrophoresis procedure will affect the species that have different charges and
therefore will attract the species according to their charges being the opposite. Species
that are positively charged will migrate towards the cathode, which is negatively charged
(because this is an electrolytic rather than galvanic cell). If the species are negatively
charged they will migrate towards the positively charged anode.
If several samples have been loaded into adjacent wells in the gel, they will run parallel
in individual lanes. Depending on the number of different molecules, each lane shows

separation of the components from the original mixture as one or more distinct bands,
one band per component. Incomplete separation of the components can lead to
overlapping bands, or to indistinguishable smears representing multiple unresolved
component. Bands in different lanes that end up at the same distance from the top contain
molecules that passed through the gel with the same speed, which usually means they are
approximately the same size. There are molecular weight size markers available that
contain a mixture of molecules of known sizes. If such a marker was run on one lane in
the gel parallel to the unknown samples, the bands observed can be compared to those of
the unknown in order to determine their size. The distance a band travels is
approximately inversely proportional to the logarithm of the size of the molecule.
Applications of electrophoresis:

Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in
restriction mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to
Northern transfer.
Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and
biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV
light and a gel imaging device. The image is recorded with a computer operated camera,
and the intensity of the band or spot of interest is measured and compared against
standard or markers loaded on the same gel. The measurement and analysis are mostly
done with specialized software.
Limitations of electrophoresis:
1. Electrophoresis Measurements Are Not Precise
Gel electrophoresis can effectively separate similar proteins with different weights (this is
a technique called Western blotting). It can separate them more precisely through a
technique known as 2d electrophoresis; this is common in proteomics.Unfortunately, all
of the measurements made from this technique are semi-quantitative at best. In order to
obtain the precise mass (weight) of proteins, mass spectroscopy must be employed after
the protein has been purified by electrophoresis. Furthermore, comparing the relative
amounts of different molecules relies on the band density (darkness) of different spots on
the gel. This method has some degree of error, and samples are usually run multiple times
to get clean results.
2. Substantial Starting Sample is Required

Electrophoresis is a technique of isolating and visually identifying different

biomolecules. This is done by passing an electrical current through the gel to separate
charged molecules of different weights. If the molecule you're interested in isn't common
enough, its band will be virtually invisible and difficult to measure.DNA and RNA can be
amplified somewhat before running electrophoresis, but it isn't practical to do this with
proteins. Therefore, a large tissue sample is needed to run these assays. This can limit the

usefulness of the technique, especially in medical analysis. It's virtually impossible to run
electrophoresis on samples from a single cell; flow cytometry and immunohistochemistry
are more commonly used to assess cell-by-cell expression of proteins. A technique called
PCR is excellent at precisely measuring tiny amounts of RNA.
3. Gel electrophoresis requires a large amount of DNA or protein to start with.
Only Certain Molecules Can Be Visualized
F

Electrophoresis is excellent at separating and identifying medium- to large-sized

biomolecules. However, many of the molecules that researchers wish to look at are
smaller; small hormones, neurotransmitters, and ions cannot be measured by
electrophoresis. This is for two reasons: they don't properly react with the electrophoresis
preparation (usually a technique called SDS PAGE) and, even if they did, they are too
small to separate properly and would rush out the bottom of the gel. These molecules are
instead measured by techniques such as RIAAs (radio immunoassays) and ELISAs
(enzyme-linked immunosorbant assay).
4. Electrophoresis is Low Throughput

Gel electrophoresis is generally low throughput, meaning it doesn't produce data

especially rapidly. Contrast electrophoresis, where you can look at a small handful of
RNA molecules at a time, with PCR (polymerase chain reaction), which can
simultaneously assess thousands of samples. Similarly, flow cytometry can take
measurements from thousands of individual cells and make complex correlations, while
electrophoresis looks at cells en masse and cannot make such fine discriminations. PCR
and flow cytometry represent massively parallel and serial processes respectively, and
both far outstrip the abilities of electrophoresis to generate research data.

III) X-ray crystallography

X-ray crystallography can locate every atom in a zeolite, an aluminosilicate.

X-ray crystallography is a tool used for identifying the atomic and molecular structure
of a crystal, in which the crystalline atoms cause a beam of incident X-rays to diffract
into many specific directions. By measuring the angles and intensities of these diffracted
beams, a crystallographer can produce a three-dimensional picture of the density of
electrons within the crystal. From this electron density, the mean positions of the atoms in
the crystal can be determined, as well as their chemical bonds, their disorder and various
other information.
Since many materials can form crystalssuch as salts, metals, minerals, semiconductors,
as well as various inorganic, organic and biological moleculesX-ray crystallography
has been fundamental in the development of many scientific fields. In its first decades of
use, this method determined the size of atoms, the lengths and types of chemical bonds,
and the atomic-scale differences among various materials, especially minerals and alloys.
The method also revealed the structure and function of many biological molecules,

including vitamins, drugs, proteins and nucleic acids such as DNA. X-ray crystallography
is still the chief method for characterizing the atomic structure of new materials and in
discerning materials that appear similar by other experiments. X-ray crystal structures can
also account for unusual electronic or elastic properties of a material, shed light on
chemical interactions and processes, or serve as the basis for designing pharmaceuticals
against diseases.
In a single-crystal X-ray diffraction measurement, a crystal is mounted on a goniometer.
The goniometer is used to position the crystal at selected orientations. The crystal is
bombarded with a finely focused monochromatic beam of X-rays, producing a diffraction
pattern of regularly spaced spots known as reflections. The two-dimensional images taken
at different rotations are converted into a three-dimensional model of the density of
electrons within the crystal using the mathematical method of Fourier transforms,
combined with chemical data known for the sample. Poor resolution (fuzziness) or even
errors may result if the crystals are too small, or not uniform enough in their internal
makeup.
Apparatus:

x-ray tubes provides a means for generating x-ray radiation in most analytical
instruments. An evacuated tube houses a tungsten filament which acts as a cathode
opposite to a much larger, water cooled anode made of copper with a metal plate on it.
The metal plate can be made of any of the following metals: chromium, tungsten, copper,

rhodium, silver, cobalt, and iron. A high voltage is passed through the filament and high
energy electrons are produced. The machine needs some way of controlling the intensity
and wavelength of the resulting light. The intensity of the light can be controlled by
adjusting the amount of current passing through the filament; essentially acting as a
temperature control. The wavelength of the light is controlled by setting the proper
accelerating voltage of the electrons. The voltage placed across the system will determine
the energy of the electrons traveling towards the anode. X-rays are produced when the
electrons hit the target metal.
Applications:

Chemistry:
properties

characterising new products and materials, investigating their

Physics: determining fundamental properties

Engineering: identifying properties of materials; stress analysis; tomography

Biology, medicine: structures of macromolecules such as proteins and DNA

Surface science: studying the structures of surfaces and interfaces under various
environmental conditions

Geology:

identification of minerals and understanding their transformations

Planetary science: studying the behaviour of atmospheric components under

extreme conditions

Pharmaceutical:
structure-based drug design, physical properties,
polymorph screening, patent applications, quality control
Nanotechnology: investigation
interdependence

of

structures,

properties

and

their

Limitations

The uncertainties introduced during the derivation of an atomic model from the
experimentally observed electron density data are not always appreciated.
Uncertainties in the atomic model can have significant consequences when this
model is subsequently used as the basis of manual design, docking, scoring, and
virtual screening efforts.
Docking and scoring algorithms are currently imperfect.
A good correlation between observed and calculated binding affinities is usually
only observed only when very large ranges of affinity are considered.
Errors in the correlation often exceed the range of affinities commonly
encountered during lead optimization.