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ELCTROPHOROESIS
Introduction
Definition
• The movement of charged particles through a colloid when subjected to an electric field
is known as electrophoresis.
• Since proteins exist as charged particle this method is widely used to the separation of
proteins in biological fluids.
Principle of Electrophoresis
Chemical species carrying an electric charged by virtue of ionization will move either to
the anode or cathode in an electrophoretic system depending on the kind of charge on a
molecule.
Apparatus
Types
• Agargel electrophoresis
o Agargel is used as a media
o Used in –
Detecting nephritic syndrome
Cirrhosis of liver
Multiple myeloma
Hepatitis
• Cellulose acetate electrophoresis
o Cellulose acetate paper is used
• Paper electrophoresis /Polyacryl Amine Gel Electrophoresis
o Gel electrophoresis is used in forensics, molecular biology, genetics,
microbiology and biochemistry
• Isoelecrtric Focussing
o The media provides a stable pH
o Isoelectric focusing (IEF) is a method of electrophoresis that separates proteins
according to their isoelectric points.
Factors affecting electrophoresis
Advantages
COLORIMETER
Introduction
Principle
• When a beam of light passed through a coloured solution it will be found that emerging
radiation is less powerful than the entering radintion.
• This is mainly due to the absorption of radiation by the light absorbing substances
present in solutions.
• This variation with change in concentration of some component solute forms the basis
for colorimetric estimation.
• Colorimeter is based on the ‘Beer – Lambert law’.
• It states that when a ray of monochromatic light is passed through an absorbing medium
its intensity decreases as the length of absorbing media increases.
• Absorbency is directly proportional to the concentration of the substance.
Types
• Visual colorimeter
o Light source – natural or white light
• Photoelectric colorimeter
o Photoelectric cell is work as eye.
Parts of Colorimeter
• Light source –
o Tungastun filament lamp – for UV range
o Mercury vapour lamps – for visible range
• Means of spectral isolation –
o Glass filters are preferred
• Cuvette –
o Round or rectangular glass cuvettes are used – range of 320 to 400
o Quartz or silicon cells - UV range
• Grating –
o It consist of large number of parallel equally spread ruled lines upon a glass or
metal surface.
• Radiant energy detectors –
o Photocells
o Phototubes
o Photomultiplier
• Galvanometer –
o Linear percentage transmission scale of 0-100
o Logarithmic absorbency scale
o Both
Application
SPECTROPHOTOMETER
Introduction
Definition
Types
• Single beam
o As the name suggests this instrument comprises a single beam of radiation
emanating from the source and travelling through the various components and
sample until ultimately reaching the detector.
o Sample absorbance is determined by measuring light intensity without the
sample in the beam and comparing it with the intensity after passing through the
sample.
• Double beam
o In this type of instrument both the blank and sample cells are placed in the
instrument at the same time
o The amount of light passing through the sample is compared with the amount
passing through the blank and the net absorbance is determined and displayed
by the instrument.
o The advantages of this system are that it is:
much faster than a single beam
very useful when a range of wavelengths are to be scanned
more stable than single beam instruments with regard to lamp intensity
drift.
Procedure