1.

ANALYTICAL BALANCE

Use: to weigh the chemicals accurately for preparation of analytical reagents.

Types: Two Pan Balance, Mono Pan Balance,Electrical Balances
Principle of Electrical Balance:
1. System equilibrium is disturbed by the weight.
2. Compensating force required to bring back equilibrium is proportional to weight.

Parts of an Electrical Balance:

1. Null detector
2. Feedback loop to control the balancing force.
3. Read out device.

Null detector Feedback loop Readout device

Working of an Electrical Balance:

Bring the instrument to zero resting point before placing the chemical for weighing
1. When weight is placed on the pan, system’s equilibrium is altered.
2. The null detector will sense the position ofbalance beam.
3. Feedback loop senses the alteration from null detector.
4. The electromagnet connected to null detector produce current which is the compensating force
to bring back equilibrium.
5. When the equilibrium is restored the null meter reads zero.
6. The magnitude of current from the null detector is proportional to sample weight.
7. The current required to produce the compensating force is converted to a digital display of the
weight.

Care and Maintenance:

1. The substance should not be weighed hot.
2. The cabinet should be closed while weighing.
3. Never weigh the substance by placing it directly on the surface of the pan.
4. Do not spill the chemicals on the pan; clean the balance after every use.
 2. CENTRIFUGE

Use:to separate particulate matter from a liquid suspension using centrifugal force.

Principle:A sample container is rotated at variable speed to generate centrifugal force with the
help of an electric motor.
The centrifugal force generated depends on:
a) mass of the particle
b) radius of rotational path from central axis
c) speed( rpm- revolutions per minute)

Parts of centrifuge:

1) Head: It is a revolving disc that contains the tube holding cups/buckets

2) Rotor: It is a high speed motor which rotates the head to create centrifugal force
3) Speedometer: controls the speed of rotation of rotor

Types of centrifuge:

1) Floor model or table top model

2) Refrigerated centrifuge
3) Ultra centrifuge

Types of centrifugal separation:

1) Differential centrifugation:This method is based on particle size and density. The

heaviest particle sediments first followed by lighter particles.

2) Density gradient centrifugation: Particles of same density but different sizes sediment
in different zones.

Care & maintenance:

1) All centrifuges should be placed on a leveled surface

2) All tubes or cups in the rotor head should be properly balanced
3) All tubes should be capped to avoid spillage
4) Centrifuge lid should be closed properly
 [Link]

Use: to analyse both coloured and colourless solutions.

Types:Spectrophotometers are classified as single-beam and double-beam.
Principle:
A beam of light is passed through a monochromator that isolates the desired region of the
spectrum to be used for measurements. Slits are used to isolate a narrow beam of the light and
improve its chromatic purity. The light next passes through an absorption cell (cuvet), where a
portion of the radiant energy is absorbed, depending on the nature and concentration of the
substance in the solution. Any light not absorbed is transmitted to a detector (photocell or
phototube), which converts light energy to electrical energy that can be registered on a meter or
recorder or digitally displayed.

Components:
Light source - Incandescent Lamps - tungsten light bulb, quartz-halogen lamp, hydrogen and
deuterium lamps, high-pressure mercury and xenon arc lamps, laser sources.
Monochromator- Devices used for spectral isolation include (1) filters (2) prisms and (3)
diffraction gratings
Cuvette - a small vessel used to hold a liquid sample to be analyzed in the light path of a
spectrometer.
Cuvets may be round, square or rectangular and are constructed from glass, silica (quartz), or
plastic.
Photodetectors - devices that convert light into an electric signal that is proportional to the
number of photons striking its photosensitive surface.
Eg. Photomultiplier tube (PMT), Photodiodes
Readout device – faster response & easier to read

LIGHT
T. LIGHT
Sample 0.01
I. LIGHT
Light Source Monochromator Cuvette PhotodetectorDisplay

Main advantages:
1. It is 1000 times more sensitive and can measure in micrograms (colorimeter only in
milligrams)
2. Wavelengths in the Ultraviolet range are also utilized and quartz cuvettes are used. (In
colorimeter UV range is not utilized and glass cuvettes are used)
3. Wavelength selection is done using Diffraction Gratings or prism (In colorimeter plane mirror
is used)
[Link] Features Colorimeter Spectrophotometer
1 Light source Tungsten lamp –emits 2 light sources
visible wavelength Tungsten lamp –emits visible wavelength
(400 to 700nm)
Deuterium lamp - emits UV (200 to 400nm)
2 Monochromator Coloured glass filter Prism or Diffraction grating
3 Cuvette Glass/Plastic Quartz(UV analysis)
4 Photocells Photodetector Plate Photomultiplier tubes
5 Uses  Only Coloured  Both coloured and colourless
solution can be solutions can be analysed
analysed  Narrow bandwidth with the use of
 Broad Bandwidth grating leading onto improved
sensitivity
 4. SEMI AUTOMATED CLINICAL CHEMISTRY ANALYSER

Use: To determine the concentration of analytes on whole blood, serum, plasma, urine and other
body fluids.
Principle:Beers Lambert’s law
Samples are applied to a tube containing reagent that produces a chemical reaction. A
light source transmits a beam of light on to a sample. The amount of absorbed light is measured
by a photodetector.
Parts:
1) Light source - Quartz halogen lamp
2) Flow cell – Quartz
3) Filter - Interference filters
4) Photodiode
5) Display
450

Halogen lamp Flow 405

Filter 490 Photodiode Display
cell
670 540

620

Working:
1) Procedures like pipetting of reagents & specimen, mixing and incubation is carried out
externally by the technician.
2) Setting of incubation temperature, zero setting, photometric reading, display,
automatic printing is carried out by the analyser.

Advantages:
1) Requires less quantity of reagent and sample.
2) Kinetic and Fixed methods can be performed accurately.
3) Data storage, data transfer to external computer and printer is possible.
4) Tests are programmed in advance.
5) Calibration facility is possible.

Disadvantage:
1) Carry over is present.
2) Time consuming.
3) Errors because of human intervention.
4) No autodilution.

Maintenance:
Flow cell rinsing should be done frequently to prevent carry over.
 5. CHEMILUMINESCENCE IMMUNOASSAY ANALYSER

Luminescence: Light emission

Chemiluminescence: Emission of light produced during a chemical reaction.

Use:to quantitate Antigen in cell lysate, plasma, urine, saliva, tissue & culture media by
detecting Glow- based chemiluminescent reactions.

Principle:
In chemiluminescence reactions, part of the chemical energy generated produces excited
intermediates that decay to ground state with emission of photons.
[A] + [B]  [◊]  [P] + Light
[A],[B] – Reactants, [◊] – Excited intermediate, [P] - Product
The emitted radiation is measured with a photomultiplier tube and the signal is related to analyte
concentration.
No excitation radiation & monochromators are needed.

Chemiluminescent labels:
Isoluminol, Luminol, Acridinium estres, Dioxetanes
Luminol + H2O2  3-Aminophthalate (excited)  3-APA + Light

Procedure:Uses microplate luminometers

o Stationary solid particles coated with Ag / Ab of interest
o Post incubation ensure intact immune complexes are formed
o Substrate is added  Light generation
o Light intensity is proportional to the amount of labeled complexes present
o Intensity of light is measured in terms of Relative Light Units (RLU)
Applications:
1. Chemiluminescence Immunoassay
2. DNA hybridization technique
3. Western blotting
4. Forensic science
5. Food analysis

Advantages:
1. Sub-picomolar detection limits
2. Speed
3. Ease of use – one step procedure
4. Simple instrumentation

Disadvantage:
Impurities produce background signal that degrades sensitivity & specificity.
 6. ELECTROPHORESIS

 Electrophoresis is a type of separation technique.

Principle:
It is based on Migration of charged particles in a liquid medium under the influence of an
electrical field.

Equipment used for the gel electrophoresis:

• Electrophoretic Apparatus-Electrophoretic tank with electrodes & power pack
• Supporting Medium - Cellulose acetate membrane, Agarose gel and
Polyacrylamide gel (PAGE)
• Buffer

Procedure:

1. Sample application
2. Adjustment of voltage or current - Direct Current
[Link] time: in minutes
(e.g. Gel electrophoresis of serum proteins - 30 mins.)
4. Electrophoresis in supporting medium: fixation, staining and destaining
5. Evaluation:
• Qualitative (standards)
• Quantitative (densitometry)

Factors affecting the migration of molecules:

Characteristics of solute:Size, charge, shape, isoelectric pH
 External Parameters:Pore size, buffer concentration, pH of the buffer, voltage, current,
temperature.
Application:
• Separation of Proteins
• Separation of Lipoproteins
• Separation of Hemoglobin
• Separation of Isoenzymes
• Separation of Nucleic acids, Nucleotides
SERUM PROTEIN ELECTROPHORESIS:
 Separation of plasma proteins by elelctrophoresis.
 Serum proteins are negative charged at alkailne pH (8.6) and they move towards the
anode.
 Albumin, small plasma protein and carring maximum negative charge moves faster than
globulins.
 Gamma globulins remain at the origin due to their large sizes.
 Beta globulins migrate between albumin and gamma globulin.

Normal pattern - contains 5 bands: albumin, α1,α2,β,γ

Condition Albumin α1 α2 β γ REMARKS

Normal Pattern Normal Normal Normal Normal Normal

Cirrhosis of Liver Decreased Normal Normal Broad Increased β-γ fusion (bridge)
Prominentα2
Nephrotic syndrome Decreased Normal Increased Normal Normal
fraction
Sharp spike –
Multiple Myeloma Decreased Normal Normal Normal Increased M band (monoclonal
band)
 7. CHROMATOGRAPHY

Chromatography is a separation technique.

Principle:
 Chromatography is a physical method of separation ofsimilar components in a mixture.
 It consists of a stationary phase and mobile phase.
 The stationary phase is a porous or solid matrix.
 Mobile phase is either liquid or gas which carries the components to be separated.
 The interaction between the stationary and mobile phase causes the separation of
components.
 The interaction can be
[Link]
[Link]
[Link] exchange
[Link] exclusion

Types of Chromatography:
1. Column chromatography
2. Paper chromatography
3. Thin layer chromatography
4. Gas liquid chromatography
5. High performance liquid chromatography

PAPER CHROMATOGRAPHY

Requirements:
 Whatmann filter paper (No. 1)
 Standard solutions: 5 mg in 1 mL of 0.1 N HCl
 Solvent system: n-Butanol, Glacial acetic acid, Water in the ration of 4:1:5
 Staining solution: Pinch of Ninhydrin in 10 ml of Acetone
 Petri dish and beaker
 Chromatography jar (Bell jar)

Procedure:
 Take half of the solvent system in a beaker and saturate the chromatography chamber and
next half in the petridish over which the Whatmann filter paper is going to be placed.
 Take a sheet of Whatman filter paper (35 cm along the length and 30 cm along the breadth).
Draw a line 5cm from the bottom of the page horizontally and draw small circles 2 cm apart
and name them.
 Then charge the spots with 7-10 µL of the solution of standard and unknown sample.
 Transfer the paper to chromatography jar and allow it to run for 16 hours.
 Take the paper out when the solvent front has ascended 80% of the paper and dry it.
 Spray the staining solution on the paper and dry it.

A measure of this mobility is called Rf value i.e. Ratio of Fronts value. It is calculated as:

Distance travelled by solute (Aminoacid)

Rf =
Distance travelled by solvent
By comparing the components with standard,we can identify them.

Uses:
 To separate closely related substances such as Carbohydrates, Amino acids, Lipids,
Proteins, Isoenzymes, Hormones.
 Paper chromatography is used for screening of disorders of Amino acids metabolism.
 8. ION SELECTIVE ELECTRODES

An ion-selective electrode (ISE), also known as a specific ion electrode (SIE), is a transducer
that converts the activity of a specific ion dissolved in a solution into an electrical potential.
The voltage is theoretically dependent on the logarithm of the ionic activity, according to
the Nernst equation.
Principle: It is based on Potentiometry. When a membrane separates two different solutions
having different ionic concentration, a potential difference is found to be present between the
surfaces of membrane. The potential is measured against a standard electrode,at a given
temperature.
TYPES OF ION SELECTIVE MEMBRANE
There are four main types of ion-selective membrane used in ion-selective electrodes (ISEs)
[Link] membranes
[Link] membranes
[Link]-exchange resin membranes
[Link] electrodes.
1. GLASS MEMBRANES
It measures H+, Na+, and Ag+.
Ex: pH glass electrode.
2. CRYSTALLINE MEMBRANES
It measures for both cation and anionof the membrane-forming substance.
Ex: Fluoride selective electrode.
3. ION-EXCHANGE RESIN MEMBRANE
It isbased on special organic polymer membranes which contain a specific ion-exchange
substance (resin)
Ex: Potassium selective electrode, based on valinomycin as an ion-exchange agent.
4. ENZYME ELECTRODES
The electrode has a "double reaction" mechanism - an enzyme reacts with a specific substance,
and the product of this reaction (usually H+ or OH−) is measured by electrode.
Ex: Glucose selective electrodes.
 9. BLOOD GAS ANALYSER
The three-channel blood analyzer (pH, PCO2, PO2) clinically provides the complete picture of
the oxygenation and acid-base status of blood.

Electrodes:
The Glass membrane & Polymer membrane electrodes are used as internal transducer for
PCO2 sensors & measuring pH respectively.
Ag/AgC1 electrode is used as the reference electrode.

Principle:
The blood sample comes into contact with specialized electrodes.
pH electrode: Measures the acidity/alkalinity of the blood.
pCo2 electrode : Measures the partial pressure of Carbon dioxide based on its reaction with a
specific solution within the electrode.
pO2 electrode : Measures the partial pressure of Oxygen based on its diffusion through a
membrane and its reaction with a specific solution within the electrode.
Each electrode converts the measured parameter into corresponding electric [Link] signal
generated by the internal electrode is logarithmic and governed by the Nernst equation

Procedure:
1. Blood specimen is presented to the sample probe.
2. Uptake of sample through the probe by a peristaltic pump.
3. Loading of the chamber with 60 to 150 pL of fluid sample.
4. Sample is allowed to reside in the chamber for thermal equilibration.
5. Completion of measurement.
6. Pump pushes the sample to waste.
7. Output is available on a display.
Under the command of the computer, calibrator gases and buffers are cycled at short
intervals through the chamber and electronic responses are continually monitored and reset to the
constant initially entered for high and low PCO2 and PO2 and high and low pH of the calibrator
materials.

MAINTENANCE OF INSTRUMENTATION:
The frequency with which maintenance should be scheduled is dependent on the number of
analyses performed in the laboratory.
Cleanliness of the sample chamber and path is especially important.
V- valve;W-waste; E1,E2,E3,E4 – electrodes.