OVERVIEW OF BIOCHEMICAL
TECHNIQUES

 

Biochemical techniques describe a
group of procedures, trials, and approaches that assist researchers to evaluate
the constituents found in existing entities and the chemical consequences
essential for life processes.

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QUALITATIVE ANALYSIS

QUANTITATIVE ANALYSIS

Predominantly inductive method used to express principle or postulates.

Predominantly
deductive method used to investigate pre-identified concepts, structures, and
postulates that sort a theory.

Statistical tests are not used in qualitative analysis.

Used of statistical tests for
quantitative analysis.

Mainly depends on ability and accuracy of the researcher.

Mainly be
determined by measuring device or tool.

Generalized in small amount.

Generalized in more amount.

 

 

 

BIOCHEMICAL TECHNIQUES

 

CHROMATOGRAPHYELECTROPHORESISSPECTROSCOPYCENTRIFUGATIONPHOTOMETRYELISA CHROMATOGRAPHY

Chromatography is a technique used to split mixtures
of constituents and converted in components. All methods of chromatography based
on the similar principle. They consist stationary and mobile phase.

PAPER CHROMATOGRAPHY

It is a technique used for separating suspended chemical
constituents by different migration rates through paper sheet.

Principle

Separation occur through liquid-liquid interaction. It
occurs between two liquid compounds and adsorption take place on paper.

Procedure

The few drops of solution of compounds which you want
to separate out is apply on one end of paper and then dip into solution then
stationary phase occur. If solvent moves to downward its descending
chromatography while move on upward motion called ascending chromatography.
Paper is removed, dried and used for spot identification.

GEL FILTRATION
CHROMATOGRAPHY

It is also known as molecular-exclusion
chromatography. it separates both small and big components.

Principle

This techniques based on molecular weight, shape and
size.

Procedure

The gel used to separate small and big molecules. The
mixture of solution for separation is apply on column and remove with buffer.
Through the pores large molecules doesn’t passed and small molecules enters in
gel beads. Through gel beads molecules were separated.

HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY (HPLC)

To separate constituents of mixture by chemical
exchanges between analyzed substance and column of chromatography.

Principle

It is a versatile technique based on several
chromatography techniques.

Procedures

Analyzed present in small volume in mobile phase.
Retardation depends on nature of analyte. The specific time in which analyte is
removed called retention time.

 

ELECTROPHORESIS

Electrophoresis is a technique that separated
macromolecules on the basis of size.

GEL-ELECTROPHORESIS

This type of electrophoresis
used to separate proteins and nucleic acid.

 

Principle

This technique separate
molecules based on size in addition to electric current.

Procedure

Gel used for media
stabilization, made wells with comb then addition of buffer take place and
sample loaded on wells then electric charges diffused in. constituents of
sample separated on the basis of their size. Components identification occur by
radio labeled or u-v spectrum.

 

 

SPECTROSCOPY

Spectroscopy refers to the immersion and radiation of light and particle
emission through matter, it depends the approaches of wavelength of emission.

MASS SPECTROSCOPY

In vaccum mass spectrometer
used to measure mass of ions .

Principle

Mainly composed of three
steps that is source, analyzer and detector.

Procedure

Analyte liquefied by a needle
on high potential. A droplets of fine spray enters in mass spectrometry then
dried by inert gas that go through analyzer in the direction of detector.
Separation of protein occur through LC.

 

CENTRIFUGATION

Centrifugation is a procedure
managed to distinct or distillate materials interrupted in a liquefied medium.
This process basically separate two mixtures.

ULTRA CENTRIFUGATION

The significant tool of
biochemical study is centrifugation, that apply high centrifugal force on
suspended particles or molecules present in solution and separation of such
matter occur on ultra-centrifugation process that is distinct in weight.

Example

Rbcs separated from plasma in
blood, mitochondrial nuclei in cells homogenate and in complex mixture from one
to another protein.

 

 

PHOTOMETRY

This is the study used to
measure of light intensity.

FLAME PHOTOMETRY
When metal is in contact with flame it is used to measure intensity
of radiant energy.

Applications

Flame photometry used to identify alkali and
alkaline earth metal.Used in soil investigation.In industrial discarded, adhesive, crystal and
petroleum. 

ELISA

ELISA established on
immunochemical ethics of antigen-antibody reaction.

 

1. The antibody beside the
protein be controlled is stable on indolent solid such as polystyrene.

2. The biological example of protein
to be projected is applied on antibody covered surface.

3. The protein antibody
complex is reacted with another protein specific antibody to which an catalyst is
covalently related. These enzymes essentially produce sooner coloured products.
Peroxidase, amylase and alkaline phosphatase are usually used.

4. After washing the uncontrolled
antibody allied enzyme, the catalyst bound to another antibody complex is analyzed.

5. The catalytic activity is resolute
by its action on substrate to the formation of color product. This linked with
concentration of protein being assessed.

Applications

ELISA mostly used to
determine small quantities of proteins and other living substances. That is normally
used for pregnancy test to detect human chorionic gonadotropin (HCG) in urine
is established on ELISA. By this, pregnancy can be revealed within few days
after outset. ELISA is also suitable for analysis of AIDS.