Various biochemistry lab instruments

VARIOUS LABORATORY
INSTRUMENTS IN BIOCHEMISTRY
Presented by:
Dr.Kusum Bala Jain
2nd
Yr Resident
Dept. of Biochemistry
GMC Kota.

CENTRIFUGE
Centrifugation is a separation technique by which
particles of different shape,size and density are separated
based
upon their sedimentation rate by spinning under the
influence of centrifugal force.(extra gravitational force)
The instrument used to hold the sample and generate the
centrifugal force is called as centrifuge.centrifuge is made
up of rotor.
.A rotor can hold the samle tube and spin along its own
axis at different speeds to generate centrifugal force.Speed

•A Urinometer is a simple piece of equipment for
determining urine specific gravity.
•A typical urinometer is composed of a float, a
weight, and a stem. The float is an air-filled glass
tube, ending in the weight and the stem.
•The weight is a bulb filled with ball bearings
embedded in a red solid, probably a glue of some
sort. The glass stem has calibrated graduations
and numbers marked off to indicate specific gravity
measurements.

•It is placed in a tube of urine, and where the
meniscus of the urine reaches displays the
specific gravity of the urine.
•Correction for temperature has to be done.
•A urinometer is typically used in medical
diagnostic labs

•It is the collective term for a set of laboratory techniques
for the separation of mixtures. The mixture is dissolved
in a fluid called the mobile phase, which carries it
through a structure holding another material called
the stationary phase.
•The various constituents of the mixture travel at
different speeds, causing them to separate.

•The separation is based on differential partitioning
between the mobile and stationary phases. Subtle
differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus
changing the separation.

•Chromatography may be preparative or analytical.

•The purpose of preparative chromatography is to
separate the components of a mixture for more
advanced use (and is thus a form of purification).
•Analytical chromatography is done normally with
smaller amounts of material and is for measuring
the relative proportions of analytes in a mixture.

Factors affecting electrophoretic
mobility
Charge
Size
Shape

COLORIMETRYCOLORIMETRY
How Do We Use This Principle?How Do We Use This Principle?
Perform a Chemical Reaction with thePerform a Chemical Reaction with the
Element to be Analyzed that Results in aElement to be Analyzed that Results in a
colored Compound of that Element aftercolored Compound of that Element after
Absorbing Light.Absorbing Light.
Measure the AmountMeasure the Amount
of Light Absorbed.of Light Absorbed.

COLORIMETRYCOLORIMETRY
1.The Chemistry Involved.1.The Chemistry Involved.
2. The Length of Light Travel.2. The Length of Light Travel.
3. The Amount (Concentration) of3. The Amount (Concentration) of
Absorbing MaterialAbsorbing Material..
The Amount of Light AbsorbedThe Amount of Light Absorbed
Is Related To:Is Related To:

CONCENTRATION CAN BE COLORIMETRICALLYCONCENTRATION CAN BE COLORIMETRICALLY
DETERMINED IF:DETERMINED IF:
1. Able to chemically develop a color with that substance and only that
substance
2.  The developed color obeys (follows) Beer’s Law over a  reasonable range
of concentrations
3. The developed color must be stable for reasonable length of time,
reproducible, and sensitive to small changes in concentration
4. All loss of transmitted light must be from absorbance by substance
measured (developed color)
5.  All of substance present in sample must be available for reaction with color
developing agent
6.  Able to measure amount of light absorbed

•A colorimeter is a device used in colorimetry. In
scientific fields the word generally refers to the device
that measures the absorbance of particular
wavelengths of light by a specific solution.
•This device is most commonly used to determine the
concentration of a known solute in a given solution by
the application of the Beer-Lambert law, which states
that the concentration of a solute is proportional to the
absorbance.

1) Wavelength selection, (2) Printer button, (3)
Concentration factor adjustment, (4) UV mode
selector (Deuterium lamp), (5) Readout, (6)
Sample compartment, (7) Zero control (100% T),
(8) Sensitivity switch, (9)ON/OFF switch[2]
The essential parts of a colorimeter are:
•a light source (often an ordinary low-voltage
filament lamp)
•an adjustable aperture
•a set of colored filters
•a cuvette to hold the working solution
•a detector (usually a photocell) to measure the
transmitted light
•a meter to display the output from the detector

Filters
•Changeable optics filters are used in the colorimeter to select the
wavelength of light which the solute absorbs the most, in order to
maximize accuracy. The usual wavelength range is from 400 to
700nanometers (nm).
•If it is necessary to operate in the ultraviolet range (below
400 nm) then some modifications to the colorimeter are needed.
In modern colorimeters the filament lamp and filters may be
replaced by several light-emitting diodes of different colors.
Cuvettes
•Main article: Cuvette
•In a manual colorimeter the cuvettes are inserted and removed
by hand. An automated colorimeter (as used in an AutoAnalyzer)
is fitted with a flowcell through which solution flows continuously.

Output
• The output from a colorimeter may be displayed
by an analogue or digital meter and may be shown
as transmittance (a linear scale from 0-100%) or
as absorbance (a logarithmic scale from zero to
infinity).
• The useful range of the absorbance scale is from
0-2 but it is desirable to keep within the range 0-1
because, above 1, the results become unreliable
due to scattering of light.
• In addition, the output may be sent to a
chart recorder, data logger, or computer.

PRINCIPLE
- Beer Lambert Law is concerned with light absorption in
relation to solution concentration and cell path length.
- It states that the intensity of a ray of monochromatic light
decreases exponentially as the concentration of the
absorbing medium increases.
- In other words, the more dissolved substance you have in
a solution, the more light that will be absorbed, and the
less light that will be transmitted through the solution.

Absorbance, Transmittance, and Reflection. A spectrophotometer measures how light interacts with
atoms or molecules in a sample.

Parts of a Spectrophotometer
• Lamp
• Prism
• Sample holder
• photomultiplier
• Display
How a Spectrophotometer Works
•White light hits grating or prism
•Light is split into colors of the rainbow
•Wavelength knob directs different colors
toward sample

How a UV Spectrophotometer Works.
Similar to a VIS spectrophotometer, the
UV spec shines ultraviolet light or visible
light on a sample, and a detector
measures the amount of light that
passes through, or is absorbed by, the
sample.
Colors of Light in the Visible
Spectrum. Humans can see
light with wavelengths of about
350 to 700 nm.

How Concentration Affects Absorbance. If a sample has twice as many molecules as another, it
can absorb twice as much light. This is true at any wavelength. It is important to know a sample’s
wavelength of maximum light absorbance, so that the difference in absorbance due to concentration is
obvious.

•A fluorometer or fluorimeter is a device used to
measure parameters of fluorescence, its intensity and
wavelength distribution of emission spectrum after
excitation by a certain spectrum of light.
•These parameters are used to identify the presence
and the amount of specific molecules in a medium.
•Modern fluorometers are capable of detecting
fluorescent molecule concentrations as low as 1 part per
trillion.
•Fluorescence analysis can be orders of magnitude
more sensitive than other techniques.
Application:
•includechemistry/biochemistry, medicine, environmental
monitoring.

Principle
Fluorescence is the emission of visible light by a
substance that has absorbed light of a different
wavelength. The emitted photon has a longer
wavelength and lesser energy.This phenomenon is
called as fluorescence.
Quantification and Characterization of fluorescent
compounds by measuring intensity of fluorescence
using fluorimeter called as fluorimetry.

Uses of flurometer
To quantify amino acids and peptides by labelling
with an extrinsic fluor,acridine orange.
For localization of enzymes in cells and metals in
metalloproteins.
For conformation alanalysis of enzymes proteins
and nucleic acid.
In cell sorting and cell counting.
To detect malignant cells
To quantify catecholamine.quinidine,and
porphyrin.

Semi Autoanalyser
Here the samples and reagents are mixed and read
manually .Based on colorimetry principle.
Disadvantages are:
More amount of sample is needed
Time consuming
Need technical expertisation

Purpose of Autoanalyzers
An autoanalyzer sequentially measures
blood chemistry through series of steps of
• mixing,
• reagent reaction and
• colorimetric measurements.

consists of different module including:
•a sampler, pump, mixing coils, optional
sample treatments dialysis, distillation,
heating, etc,
•a detector, and data generator.
•Most continuous flow analyzers depend on
color reactions using a flow through
colorimeter

Principle of operation
In Segmented Flow Analyzers (SFA), the sample is
mixed with small reproducible volumes of the required
reagents
air bubbles are introduced into the flow, creating
about 20 - 100 segments of liquid for each sample
The sample / reagent mixture flows through mixing
coils (heated coils)  a color proportional to the
amount of analyte in each sample is developed
The samples with developed color flow through a
colorimeter to measure the color

It consists of
Sampler:
 Aspirates samples, standards, wash solutions into the
system
Proportioning pump:
 Mixes samples with the reagents so that proper
chemical color reactions can take place, which are then
read by the colorimeter
Dialyzer:
 The purpose of a dialyzer is to separate the analyte
from interfering substances such as protein, whose
large molecules do not go through the dialysis
membrane but go to a separate waste stream
 The analyte infuses through the diaphragm into a
separate flow path going on to further analysis

It consists of
Heating bath:
 Controls temperature (typically at 37 °C), as temp
is critical in color development
Colorimeter:
 Monitors the changes in optical density of the fluid
stream flowing through a tubular flow cell. Color
intensities proportional to the substance
concentrations are converted to equivalent
electrical voltages .
Recorder:
 Displays the output information in a graphical
form.

•It is a very sensitive in vitro assay technique used to
measure concentrations of antigens (for example,
hormone levels in the blood) by use of antibodies. As
such, it can be seen as the inverse of a
radiobinding assay, which quantifies an antibody by use
of corresponding antigens.
•Although the RIA technique is extremely sensitive and
extremely specific, requiring specialized equipment, it
remains among the least expensive methods to perform
such measurements. It requires special precautions and
licensing, since radioactive substances are used.
Radioimmunoassay (RIA)

Method:
To perform a radioimmunoassay, a known quantity of
an antigen is made radioactive, frequently by labeling it
with gamma-radioactive isotopes of iodine, such as
I-125, attached to tyrosine.
This radiolabeled antigen is then mixed with a known
amount of antibody for that antigen, and as a result,
these two specifically bind to one another. Then, a
sample of serum from a patient containing an unknown
quantity of that same antigen is added.
This causes the unlabeled (or "cold") antigen from the
serum to compete with the radiolabeled antigen ("hot")
for antibody binding sites.

As the concentration of "cold" antigen is
increased, more of it binds to the antibody,
displacing the radiolabeled variant, and reducing
the ratio of antibody-bound radiolabeled antigen
to free radiolabeled antigen.
The bound antigens are then separated from
the unbound ones, and the radioactivity of the
free antigen remaining in the supernatant is
measured using a gamma counter

Applications of
Radioimmunoassays
Endocrinology
Insulin, HCG, Vasopressin
Detects Endocrine Disorders
Physiology of Endocrine Function
Pharmacology
Morphine
Detect Drug Abuse or Drug Poisoning
Study Drug Kinetics

Applications of
Radioimmunoassays
Epidemiology
Hepatitis B
Clinical Immunology
Antibodies for Inhalant Allergens
Allergy Diagnosis
Oncology
Carcinoembryonic Antigen
Early Cancer Detection and Diagnosis

CHEMILUMINESCENCE
Emission of light with limited emission of heat
(luminescence), as the result of a chemical reaction.
[A] + [B] [◊] [→ → Products] + light
[A], [B]: reactants
[◊]: excited intermediate

For example, if [A] is luminol and [B] is hydrogen peroxide
in the presence of a suitable catalyst we have:
luminol + H2O2 →3-APA[◊] →3-APA + light
Where:
3-APA is 3-aminophthalate
3-APA[◊] is the excited state producing light as it decays
to a lower energy level.

CHEMILUMINISCENCE
Luminol and peroxidase
before adding H2O2
Chemiluminiscence after
addition H2O2

68
What is
AAS ?Atomic absorption spectroscopy is a
quantitative method of analysis that is
applicable to many metals and a few
nonmetals.
The technique was introduced in 1955 by
Walsh in Australia
IntroductionIntroduction

69
PRINCIPLEPRINCIPLE
A much larger number of the gaseous metal atoms will
normally remain in the ground state.
These ground state atoms are capable of absorbing radiant
energy of their own specific resonance wavelength.
If light of the resonance wavelength is passed through a flame
containing the atoms , then part of the light will be absorbed.
The extent of absorption will be proportional to the number of
ground state atoms present in the flame.

70
What is AAS ?
• An atomic absorption spectrophotometer
consists of a light source, a sample
compartment and a detector.
Light SourceLight Source DetectorDetector
SampleSample
CompartmentCompartment

72
Fuel and oxidant
flame
 Air – acetylene
Air- propane
Air- hydrogen
 Nitrous oxide – acetylene
Auxiliary
oxidant
Fuel

77
InstrumentationInstrumentation
Line
source Monochromator Detector
Read-outNebulizer
Schematic diagram of a flame spectrophotomer
Atomization

Background
The impact of a stream of high energy electrons
causes the molecule to lose an electron forming a
radical cation.
A species with a positive charge and one unpaired
electron
+ e
-
C H
H
H
H H
H
H
HC + 2 e
-
Molecular ion (M+
)
m/z = 16

BackgroundThe impact of the stream of high energy electrons
can also break the molecule or the radical cation
into fragments.
(not detected by MS)
m/z = 29
molecular ion (M
+
) m/z = 30
+ C
H
H
H
+ H
HH C
H
H
C
H
H
H C
H
H
C
H
H
H C
H
H
+ e
-
H C
H
H
C
H
H
H
m/z = 15

Background
Only cations are detected.
Radicals are “invisible” in MS.
The amount of deflection observed depends on the
mass to charge ratio (m/z).
Most cations formed have a charge of +1 so the amount
of deflection observed is usually dependent on the mass
of the ion.

Fragmentation Patterns
The impact of the stream of high energy electrons
often breaks the molecule into fragments, commonly
a cation and a radical.
Bonds break to give the most stable cation.

Mass Spectrometer can be combined with gas
chromatography to analyze mixtures of compounds.
GC separates the components of the mixture.
Each component is analyzed by the Mass
Spectrometer.

pH meter
It is an electronic device used for measuring the pH which is
the concentration of Hydrogen ions in an aqueous solution.
The pH meters work in liquids though special probes are
sometimes also used to measure the pH of semi-solid
substances.
Typical pH meter consists of a special measuring probe
(a glass electrode) connected to an electronic meter that
measures and displays the pH reading.

pH Meter
A sample is placed in a cup and the glass probe at the
end of the retractable arm is placed in it.
The probe is connected to the main box.
There are two electrodes inside the probe that
measure voltage.
One is contained in liquid with fixed pH.
The other measures the acidity of the sample through
the amount of H+ ions.

pH Meter
A voltmeter in the probe measures the difference
between the voltages of the two electrodes.
The meter then translates the voltage difference into
pH and displays it on the screen.
Before taking a pH measurement the meter must be
calibrated using a solution of known pH.

Effect of Temperature and Buffers
Temperature compensation is contained within
the instrument because pH electrodes are
temperature sensitive.
Temperature compensation only corrects for the
change in the output of the electrode, not for the
change in the actual solution.
Buffers are solutions that have constant pH values
and the ability to resist changes in pH.
They are used to calibrate the pH meter.

The thermal cycler (also known as a thermocycler, PCR
machine or DNA amplifier) is a laboratory apparatus most
commonly used to amplify segments of DNA via
the polymerase chain reaction (PCR).
 Thermal cyclers may also be used in laboratories to
facilitate other temperature-sensitive reactions, including
but not limited to restriction enzyme digestion or rapid
diagnostics.
 The device has a thermal block with holes where tubes
holding the reaction mixtures can be inserted. The cycler
then raises and lowers the temperature of the block in
discrete, pre-programmed steps.

HPLC
Chromatography is a physical process whereby
components ( solutes ) of a sample mixture are
separated by their differential distribution between
stationary & mobile phases .
Planar & column are two basic forms of
chromatography .
High performance liquid chromatography is a form of
column chromatography .

contd
 During column chromatography process mobile phase
carries the sample through the column containing
stationary phase .
 As the mobile phase flows through the stationary
phase the solutes may
1) Reside only on stationary phase ( no migration ) ,
2) Reside only in the mobile phase ( migration with
mobile phase ) ,
3) Distribute between 2 phases ( differential migration)
The basis of all forms of chromatography is partition or
distribution coefficient ( Kd )

Principle of HPLC
The limit to the length of the column is due the problem
of peak broadening .
The number of theoretical plates is related to the surface
area of the stationary phase therefore smaller the
particle size of the stationary phase , the better is the
resolution.
The Smaller the paritcle size , the greater is the
resistance to flow of the mobile phase .

contd
The resistance in flow causes back pressure in the
column that is sufficient to damage the matrix structure
of the stationary phase .
The new smaller particle size stationary phases that can
withstand high pressures causes dramatic development in
the column chromatography .

Instrumentation
The increased resolution achieved in HPLC
compared to classical chromatography is primarily
the result of adsorbents of very small particle size
( less then 20µm )& large surface areas .
The smallest gel beads used in gel exclusion
chromatography are superfine grade with
diameters of 20-50µm .
A combination of high pressure & adsorbents of
smaller size leads to high resolution power &
short analysis time in HPLC .

 (1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4)
Mixing vessel for delivery of the mobile phase, (5) High-pressure
pump, (6) Switching valve in "inject position", (6') Switching valve in
"load position", (7) Sample injection loop, (8) Pre-column (guard
column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data
acquisition, (12) Waste or fraction collector.

contd
Repeated application of highly impure samples such as
sera , urine , plasma or whole blood are preferably
deproteinated because they decrease the resolving power
of the column .
To prevent the above problem a guard column is
frequently installed between the injector & the analytical
column .

HPLC with Mass spectrometer
Narrow-bore columns (1-2 mm) are used for in this
application .
 Liquid chromatography-mass spectrometry (LC-MS,
or alternatively HPLC-MS) is an analytical chemistry
technique that combines the physical separation
capabilities of liquid chromatography (or HPLC) with the
mass analysis capabilities of mass spectrometry.

Application of HPLC
HPLC has had big impact on separation of oligopeptides
& proteins .
FPLC a modified version useful in separation of proteins .
HPLC coupled with electrochemical detector is useful in
assay of catecholamines ,vitamins (AD&E ,niacin ,
thiamine) & antioxidants .
HPLC has role in quantification of various hemoglobins
in hemoglobinopathies .
HPLC coupled with MS is useful in measuring cortisol in
blood & saliva .

contd
HPLC is useful in cytokine measurement .
Useful in assay of HbA1c .
Useful in assay of fructosamine .
5 – hydroxy idole acetic acid & serotonin can be assayed.
The pharmaceutical industry regularly employs Reverse
Phase HPLC to qualify drugs before their release.
Assay of plasma & urinary catecholamines , plasma &
urinary metanephrines

contd
For diagnosis of different porphyrias .
Thyroxine , uric acid .
Nucleic acid analysis, oliginucleotides , steroids , amino
acids , serotonin , measurement of isoenzymes .

Various biochemistry lab instruments

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