BIOCHEMICAL sample solution and the physical properties of

BIOCHEMICAL TECHNIQUES

 

“Biochemical analysis techniques are
defined as the methods, assays, and procedures that allow scientists to reveal
the substances present in living organisms and the chemical reactions
underlying life processes.”

 

It is a
rapidly developing field of science and is a crucial part of modern drug
discovery and research. This field emphasis on the chemical composition of
living systems utilizing certain experimental techniques and computational
simulations.

To carry out
biochemical analysis of a biomolecule in a biological system, it is essential
to design an approach to detect the biomolecule and extract it in pure form.
For the purification of a biomolecule various techniques such as
centrifugation, electrophoresis, precipitation, chromatography etc. are used.

 

BIOCHEMICAL
TECHNIQUES

Ø Centrifugation

Ø Microscopy

Ø Spectrophotometry

Ø Chromatography

Ø Electrophoresis

Ø Flame Photometry

Ø Fluorometry

Ø Spectroscopy

Ø ELISA

 

CENTRIFUGATION

Centrifugation is
a technique employed for the separation of particles from a solution according
to their size, shape, density, viscosity of the medium and rotor speed.

CENTRIFUGE

A centrifuge
is a device that separates particles from a solution by using a rotor. In
biology, the particles are usually cells, subcellular organelles, or large molecules.

WORKING

The particles suspended in a liquid medium
are put in a centrifuge tube
and the tube is then settled in a rotor
and spun at a specified speed. The centrifuge grips the top of the tubes and
the bottom is free to angle out. As it spins, a centrifugal force is exerted to
every particle in the sample. The particles will sediment at the speed
proportional to the centrifugal force exerted on it. The viscosity of the
sample solution and the physical properties of the particles also influence the
sedimentation rate of each particle.

Thus, by
centrifugation red cells from plasma of blood, nuclei from mitochondria in cell
homogenates, and one protein from another in complex mixtures can be separated

TYPES OF CENTRIFUGES

Microcentrifuges

Small Benchtop Centrifuges

General Purpose Centrifuges

Large Capacity Centrifuges

Superspeed Centrifuges

Ultracentrifuges

 

SPECTROPHOTOMETRY

Spectrophotometry
is a technique used to measure the amount of light absorbed by a chemical
substance by estimating the intensity of light as a beam of light
passes through sample solution.

PRINCIPLE

The
basic principle is that each compound absorbs or transmits light over a certain
range of wavelength. The spectrophotometer measures that amount if light
absorbed in order to estimate the concentration of an unknown solution.

INSTRUMENTATION

A
spectrophotometer is an instrument that estimates the intensity of light
absorbed as it transmits a sample solution. It consists of two devices, a
spectrometer and a photometer.

·       
Spectrometer:

A
spectrometer is a device that produces, typically scatters and estimates
light. It produces a desired range of wavelength of light. First a
collimator emits a straight beam of light that transmits through a
monochromator to disperse it into several component wavelengths referred to as
spectrum. Then a wavelength slit passes only the required wavelengths.

·       
Photometer:

A photometer is the
photoelectric detector that measures the amount of light. After
the desired range of wavelength of light transmits through the sample
solution in cuvette, the photometer detects the intensity of light that is
absorbed and then conveys a signal to a galvanometer or a digital display
provided.

TYPES
OF SPECTROPHOTOMETER

A
spectrophotometer can be classified into two different types depending on the
range of wavelength of light source

UV-visible spectrophotometer: 

It uses
light over the ultraviolet range (185 – 400 nm) and visible range (400 –
700 nm) of electromagnetic radiation spectrum.

IR spectrophotometer: 

It uses light over the infrared range (700 – 15000 nm) of
electromagnetic radiation spectrum.

 

CHROMATOGRAPHY

Chromatography is a technique that utilizes a group
of methods  for separating the components
of very small quantities present in complex mixtures on the basis of the relative
amounts of each component distributed between a moving fluid streams, called
the mobile phase, and a contiguous stationary phase. The mobile phase may be
either a liquid or a gas, while the stationary phase is either a solid or a
liquid.

HISTORY

The chromatography technique was
introduced early in the twentieth century when a Russian botanist Mikhail
Tswett used a column packed with calcium carbonate to separate plant pigments.
It is one of the most important techniques in environmental analysis.

Principle

The principle of chromatography
is based on the fact that a substance that is in contact with two immiscible
phases, moving and stationary, will equilibrate among them. A reproducible part
will separate into each phase, according to the relative affinity of the
substance for each phase. A substance which has affinity for the moving or mobile phase will be moved rapidly
through the system. A material which has a stronger affinity for the stationary phase will spend more time
immobilized in that phase, and will require longer time to pass through the system.

TYPES
OF CHROMATOGRAPHY

Paper chromatography

Thin layer chromatography (TLC)

Liquid column chromatography

Size exclusion chromatography

Ion-exchange chromatography

Affinity chromatography

Gas chromatography

 

 

Electrophoresis

Electrophoresis is a chromatography technique by which a mixture of charged
molecules is differentiated on the basis of size when placed in an electric
field. This technique is frequently performed in the lab to differentiate
charged molecules, such as DNA, on the basis of their size.

PRINCIPLE

During
electrophoresis charged molecules move through a gel
as an electric current is applied across the gel so that one end of the gel is
a positively charged while the other end is a negatively charged. The movement
of charged molecules is referred to as migration. Molecules migrate towards the
opposite charge. A molecule with a negative charge will move towards the positive
end and the molecule with positive charge will move towards the negative end.
Smaller molecules move through the gel more rapidly and therefore move farther
than larger fragments that move more slowly and therefore travel a shorter
distance. Eventually the molecules are differentiated according to size.

 

WORKING

 To perform electrophoresis DNA samples are loaded into wells
at one end of a gel and an electric current is applied to pull them through the
gel. As DNA fragments are negatively charged they move towards the positive electrode.
When a gel is stained with a DNA-binding dye, the DNA fragments can be visualized
as bands, each presenting a
group of same-sized DNA fragments.

 

SPECTROSCOPY

Spectroscopy is the study of the interaction of
electromagnetic radiation in all its forms with matter. It can be more
specifically defined as the study of the interactions between particles such as
electrons, protons, and ions, as well as their interaction with other particles as
a function of their collision energy.

PRINCIPLE

When a beam of white light strikes a triangular prism it is
differentiated into its various components (ROYGBIV) which is called a
spectrum. The optical system which enables production and visualizing of the
spectrum is called a spectroscope. There are many other forms of light which
are invisible to the human eye and spectroscopy is extended to cover all these.

 

APPLICATIONS

Spectroscopic analysis has been necessary in the development
of the most fundamental theories in physics, including quantum mechanics, the special
and general theories of relativity, and quantum electrodynamics. It has been a
vital tool in developing scientific understanding of the electromagnetic force
and strong and weak nuclear forces.

 

FLAME
PHOTOMETRY

 

Flame
Photometry is a branch of spectroscopy in which the species studied in the
spectrometer are in the form of atoms.

Flame
Photometer is used in inorganic chemical analysis to analyze the concentration
of certain metal ions such as sodium, potassium, calcium and lithium. It is based
on measurement of amount of the light emitted when a metal is introduced into
flame. The wavelength of color determines the element qualitatively. The color
intensity determines the element quantitatively.

PRINCIPLE

The general
principle of Spectroscopy is based on the fact that “Matter absorbs light
at the same wavelength at which it emits light.” Atoms of elements when subjected
to hot flame its orbital electrons absorbed Specific quantum of thermal energy and
become unstable at high energy level. As the excited electron move back to
ground state they release energy as photons which is of particular wavelength.

INSTRUMENTATION

A burner
that supplies flame and can be kept in a constant form and at a constant
temperature. There is a Nebulizer and Mixing Chamber which helps to take the
homogeneous solution of the substance into the flame at a steady rate. The
optical system consists of three parts: convex mirror, lens and filter. The Photo
Detector detects the emitted light and measure the intensity of radiation
emitted by the flame. Therefore the emitted radiation is converted to an electrical
signal with the aid of photo detector. The given electrical signals are
directly proportional to the intensity of light.

 

FLUORIMETRY

Fluorimetry is a spectroscopic analytical technique
for the detection and measurement of fluorescence in compounds such as cells,
proteins, or nucleotides that are labeled with fluorescence agents.

PRINCIPLE

Fluorescence
is a phenomenon of emission of radiation when the molecules are excited by
radiation at certain wavelength. Molecule contains electrons that may be
present in bonding molecular orbital which is called as highest occupied
molecular orbital (HOMO).It has least energy and is more stable. When the
molecules absorbs radiant energy from a light source, the bonding electrons may
be excited to anti bonding molecular orbital that has more energy and hence is
less stable. As the molecule falls back from the excited state to the ground
state it emits radiation that can the measured by fluorimeter as fluorescence.

INSTRUMENTATION

A fluorimeter is a spectrometer that is very similar to the
spectrophotometer, and contains a light source and a filter or monochromator to
set a specific group of excitation wavelengths, which are then targeted into a
sample. The light produced from the sample is then transmitted through another
filter or monochromator which determines the emission wavelength of interest as
well as eliminating most of the excitation light, prior to the measurement by a
detector.

Types of Fluorimeter

There are two basic types of fluorimeters:

·       
Filter fluorimeter

·       
Spectrofluorimeter

 

 

ELISA

The Enzyme-Linked Immunosorbent Assay (ELISA) is a technique
used to observe antibodies or infectious agents in a sample. ELISA test
diagnoses infections such as HIV (human immunodeficiency virus) and some allergic
diseases like food allergies.

TYPES OF ELISA

There are four types or kinds of
ELISA tests:

Direct ELISA

Direct ELIZA is the attachment of an
antigen to a polystyrene plate proceeded by an enzyme-labeled antibody that can
react with the antigen and a substrate that can be measured.

Indirect ELISA

Indirect ELIZA is the attachment of
an antigen to a polystyrene plate proceeded by an unlabeled or primary antibody
proceeded by an enzyme-labeled antibody that can react with both the primary
antibody and substrate.

·        
Sandwich ELISA

During a sandwich ELIZA a captured
antibody is bound to the polystyrene plate and an antigen is introduced that
specifically attaches or captures the antigen. A second antibody that is also
specific for the antigen but not the same as the capture antibody is introduced
and “sandwiches” the antigen. This second antibody is then proceeded by
an enzyme-labeled antibody specific for the second antibody that can react with
a substrate that can be measured.

·        
Competitive ELISA

This test involves the addition of competing antibodies or
proteins as the second antibody is added. This results in a decrease in the substrate
signal that is produced. This test gives highly specific results.