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INTRODUCTION:      

Biochemical analysis techniques refer
to a set of methods, assay,
and procedures that enable scientists to study the substances found in living
organisms and the chemical reactions essential life processes. The most complicated of
these techniques are reserved for specialty research and diagnostic laboratories,
while simplified sets of these techniques are used in such common procedures as
testing for banned drug abuse in competitive athletic events and monitor of
blood sugar by diabetic patients.

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LIST OF BIOCHEMICAL TECHNIQUES:

Biochemical
lab methods, systematic and analysis methods.

·        
Spectroscopic
methods

·        
Electrophoretic
techniques

·        
Chromatography

·        
Calorimeter

·        
Photometery

·        
Nuclear
magnetic resonance

·        
Centrifugation

·        
ELISA

·        
DNA
cloning and sequencing

·        
use
of radioisotopes

·        
immunoassay
methods

1.CHROMATOGRAPHY:

Chromatography is one of the most
helpful and accepted tools of biochemistry. It is an analytical technique
dealing with the separation of closely associated compounds from a mixture.
These consist of proteins, peptides, amino acids, lipids, carbohy­drates,
vitamins and drugs.

Principles and categorization

Chromatography 
usually consists of a mobile phase and a stationary phase.

·      
MOBILE PHASE:

The mobile phase refers to the combination
of substances(to be seperated), dissolve in a liquid or a gas.

·      
STATIONARY PHASE:

The stationary stage is a porous
solid matrix through which the sample contained in the mobile phase percolates.

The contact between the mobile
and stationary phases results in the separation of the compounds from the
mixture. These interactions consist of the physicochemical principles for
example adsorption, partition, ion-exchange, molecular sieving and affinity.

The interaction between stationary
phase and mobile phase is frequently employed in the classification
chromatography e.g. partition, adsorbtion, ion- exchange. More, the categorization
of chromato­graphy is also base either on the nature of the stationary phase (paper,
thin layer ,column), or on the nature of both mobile and stationary phases (gas-
liquid chromatography).

 

 

2.ELECTROPHORESIS:

The movement of charged particles
(ions) in an electric field resulting in their migration towards the oppositely
charged electrode is called as electrophoresis. Molecules with a net positive
charge (cations) move towards the negative cathode whereas those with net
negative charge (anions) transfer towards positive anode. Electrophoresis is a
widely used analytical method for the separation of biological molecules for
example plasma proteins, lipoproteins and immunoglobulin’s.

TYPES OF ELECTROPHORESIS:

·        
Zone of electrophoresi

·        
Immunoelectrophoresis:

·        
Isoelectric focusing:

3.PHOTOMETERY:

Photometry broadly deals with the learn of the phenomenon of
light absorption by particles in solution. The specificity of a compound to take
up light at a particular wavelength (monochromatic light) is exploited in the
laboratory for quantitative measurements.

COLORIMETER:

Colorimeter (or photoelectric
colorimeter) is the tool used for the measurement of coloured substances. This apparatus
is operative in the observable range (400-800 nm) of the electromagnetic
spectrum of light. The functioning of colorimeter is based on the principle of
Beer-Lambert law.

The colorimeter, in general
contain light source, filter sample holder and detector with display (meter or
digital). A string lamp usually serves as a light source. The filters allow the
passage of a minute range of wave length as incident light.

4.SPECTROPHOTOMETERY:

The spectrophotometer primarily differ
from colorimeter by covering the ultraviolet region (200- 400 nm) of the
electromagnetic spectrum. additional the spectrophotometer is more complicated
with numerous additional devices that eventually raise the sensitivity of its
operation several fold when compare to a colorimeter.

A precisely selected wavelength ( 234 nm or 610 nm) in both
ultra violet and visible range can be use for measurements. In place of glass
cuvettes (in colorimeter), quartz cells are used in a spectro­photometer. The
spectrophotometer has similar basic parts describe for a colorimeter 

5.ULTRACENTRIFUGATION:

Ultracentrifugation is an indensable
instrument for the isolation of subcellular orgaelles proteins and nucleic acid.
as well, this technique is also in use for the purpose of molecular weights of
macromolecules. The rate at which the sedimentation occur in ultracentrifgation
primarily based on the mass and shape of the particles or macromolecules (i.e.
on the molecular weight). It is expressed in terms of sedimentation coefficients).

6.CENTRIFUGATION:

centrifugation is the use of the centrifugal forces generated
in a spinning rotor to divide biological particles,it includes cells, viruses,
sub?cellular organelles, macromolecules (principally proteins and nucleic
acids) and macromolecular complexes (such as ribo nucleoproteins and lipoproteins).
The three mainprocedures of separation are differential pelleting, rate?zonal
centrifugation and isopycinic centrifugation. The first two methods separate
particles primarily on the basis of volume while isopycinic centrifugation
separates particle on the basis of their density. The choice of centrifugation technique
based on the nature of the particles and often above one separation technique
is compulsory for example, membrane fractionation often involves first making
an enriched fraction from a cell homogenate by differential pelleting
followed by isopycinic

7.NUCLEAR MAGNETIC RESONANCE:

Nuclear Magnetic Resonance
(NMR) spectroscopy is an analytical chemistry method used in quality control and research for determining the content
and purity of a test with its molecular
structure, centrifugation to attain purified fractions.

8.MASS SPECTROMETRY:

Mass
spectrometry is a useful analytical technique used to quantify known materials,
to identify unidentified compounds in a sample, and to elucidatethe structure
and chemical properties of dissimilar molecules. The whole process includes the
change of the sample into gaseous ions, with or not including fragmentation,
which are then characterizeby their massto charge ratios (m/z) and relative abundances.

This method mainly
studies the effect of ionizing energy on molecules. It based upon chemical reactions
in the gas phase wherein sample molecules are consumed during the formation of
ionic and neutral species.

PRINCIPLE:

The initial step in the mass spectrometric analysis of compounds is the manufacturing
of gas phase ions of the compound, mostly by electron ionization. This
molecular ion undergoes fragmentation. Each primary product ion derived from
the molecular ion, in turn, undergoes fragmentation, and so on. The ions are
separated in the mass spectrometer in accordance with their mass-to-charge
ratio, and are detected in proportion to their great quantity A mass spectrum
of the molecule is thus formed. It displays the result in the form of a plot of
ion abundance versus mass-to-charge ratio. Ions give information concerning the
nature and the structure of their precursor molecule. In the spectrum of a pure
compound, the molecular ion, if present, appears at the highest value of m/z
(followed by ions containing heavier isotopes) and gives the molecular mass of
the compound.

9.ELISA:

ELISA is depend
on the immunochemical principles of antigen- antibody effect 

 

1.The
antibody in opposition to the protein to be determined is set on an inert solid
for example polystyrene.

2. The biological sample contain
the protein to be estimatedis useful on the antibody coated surface.

3. The protein antibody complex
is then reacted with a second protein specific antibody to which an enzyme is
covalently related. These enzymes must be easily assayable and make preferably
coloured products. peroxidase,amylase and alkaline phosphaase are normally
used.

4. Later than washing the unbound
antibody linked enzyme, the enzyme bound to the second antibody complex is
assayed.

5. The enzyme activity is
identifies by its action on a substrate to form a product (usually coloured).
This is related to the concentration of the protein being estimated

APPLICATIONS:

ELISA is widely used for the determination
of small quantities of proteins (hormones, antigen, antibodies) and other
biological substances. The most commonly used pregnancy test for the detection of
human chorionic gonadotropin(hCG) in urine is based on ELISA. By this test,
pregnancy can be detected within few days after conception. ELISA is also
useful for the diagnosis of AIDS.

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