Various biochemistry lab instruments

this presentation is covering every biochemistry lab instrument we are using in brief

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

2. CENTRIFUGE

3. 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
of rotor spin denoted as rpm.

5. Urinometer

6. •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.

7. •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

9. •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.

10. •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. 

11. ELECTROPHORESIS

15. Factors affecting electrophoretic
mobility
Charge
Size
Shape

16. COLORIMETER

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

18. 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) of 3. The Amount (Concentration) of
Absorbing MaterialAbsorbing Material..
The Amount of Light AbsorbedThe Amount of Light Absorbed
Is Related To:Is Related To:

19. 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

20. •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.

21. 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

22. 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.

23. 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.

24. SPECTROPHOTOMETER

26. 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.

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

30. 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

31. 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.

32. 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.

33. FLUROMETER

34. •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.

35. 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.

39. 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.

40. SEMI AUTOANALYZER

41. 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

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

44. 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

45. 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

46. 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

47. 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.

48. Block diagram

49. ELECTROLYTE ANALYZER

55. •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)

56. 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.

57. 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

59. Radioimmunoassay Procedure

60. 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

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

62. 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

63. 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.

65. CHEMILUMINISCENCE
Luminol and peroxidase
before adding H
2
O
2
Chemiluminiscence after
addition H
2
O
2

68. 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. 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. 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. 72
Fuel and oxidant
flame
 Air – acetylene
Air- propane
Air- hydrogen
 Nitrous oxide – acetylene
Auxiliary
oxidant
Fuel

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

83. 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
-
CH
H
H
H H
H
H
HC + 2 e
-
Molecular ion (M
+
)
m/z = 16

84. 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
HHC
H
H
C
H
H
HC
H
H
C
H
H
HC
H
H
+ e
-
HC
H
H
C
H
H
H
m/z = 15

86. 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.

87. 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.

91. 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.

93. ARTERIAL BLOOD GAS ANALYZER

97. pH meter

98. 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.

99. 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.

100. 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.

101. 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.

102. THERMAL CYCLER

103. 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.

104. HbA1c Analyzer

106. 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 .

107. 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 )

108. 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 .

109. 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 .

110. 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 .

111. (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.

113. 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 .

114. 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.

115. 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.

116. 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 .

117. 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

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