INSTRUMENTS

ELCTROPHOROESIS

Introduction

• It is the most common method of analyzing the plasma proteins.

• It refers to the migration of charged particles in media under the influence of electric
field.
• It is discovered by Tisselius in 1937.

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

• Electrophoresis process tank – to hold buffer filled with electrodes.

• Power pack – to supply electricity at constant current and voltage
• Buffer – to ensure effective separation of mixture of proteins
• The pH, ionic strength, and nature of the buffer may be varied according to the proteins
to be separated.

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

• Rate of migration depends on

• Net charge on the particle
• Mass and shape of the particle
• pH of the medium
• Strength of the electric field properties
• Properties of the supporting medium
• Temp
• Dilution of sample

Advantages

• Minimize zone spreading

• Improve heat dissipation
• Shorter separation times

COLORIMETER

Introduction

• Colorimeter is an instrument used for the measurement of selective absorption of

radiation in solution when coloured light is passed through.
• This device was invented by Jan Szczepanik.

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

This type of colorimeter has a wide range of applications,

• Including laboratory research,

• Environmental analysis of water quality,
• Analysis of soil components,
• Monitoring of hemoglobin content in the blood and
• Analysis of chemicals used in various industrial setting

SPECTROPHOTOMETER

Introduction

• Spectrophotometer is a modified and advanced instrument of colorimeter for

measurement of light intensity.
• Important features of spectrophotometers are spectral bandwidth and linear range of
absorption measurement.

Definition

• A spectrophotometer is a photometer (a device for measuring light intensity) that can

measure intensity as a function of the color (or more specifically the wavelength) of
light.
• An instrument used for measuring the transmission or reflection of light by comparing
various wavelengths of the light.

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.

The components of spectrophotometer

• Source of radiant energy

o A tungsten filament lamp – visible light source
o Hydrogen and Deuterium discharge lamps – UV light
• Slit
o Entrance and Exit – these brings uniformity in beam of light
• Wave length selector –
o for the selection of a particular wavelength from the broad band radiation
 monochormator
 grating monochromator
 prisms
• Cuvette
o It is a receptacle in which a solution is placed for a spectrometric measurement.
o It is also known as cell.
o Glass cuvette used for 320 to 920 nm range
o Quartz or Silica used for below 302 nm
o Types – square & circular
o Uses – to estimate the concentration of blood sugar, blood urea, uric acid and
serum creatinine.
• Detector
o Used for the measurement of intensity of the radiation passing from the
sample.
• Read out device
o Galvanometer – read in either transmittance or absorbance units

Procedure

The blank and reference – inferred and adjusted to 100% transmission

A standard cuvette is prepared

Sample is inserted and readings are noted

Formula for Calculation

𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕 𝑶𝑶𝑶𝑶 𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄.𝒐𝒐𝒐𝒐 𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔
× 100
𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔 𝑶𝑶𝑶𝑶 𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗 𝒐𝒐𝒐𝒐 𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃 𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕