BIOCHEMISTRY EQUIPMENT

MODULE 2
 Automated clinical analyzers – Biochemistry
analyzers
 Clinical chemistry analyzers run assays on clinical samples
such as blood serum, plasma, urine, and cerebrospinal fluid to
detect the presence of analytes relating to disease or drugs.
How does a clinical analyzer work?
• Analyzers are highly automated to maximize throughput, to
improve user safety from biohazards, and to diminish the risk
of cross-contamination.
• Samples are loaded into the machine and tests are
programmed by the user. A probe measures an aliquot of
sample and places it into a reaction vessel. The RX daytona from Randox is a compact, fully automated
benchtop clinical chemistry analyzer suitable for use in small- to
• Reagents are added from an on-board refrigerated supply. medium-throughput laboratories.

Incubation time is allowed, if required; then photometric or

ion-selective electrode (ISE) testing determines the
concentration of analyte.
• Results are displayed on screen or sent to a printer or
computer.
BIOCHEMISTRY ANALYSER
 BIOCHEMISTRY ANALYSER
 The Clinical Biochemistry Analyzer is an instrument that uses the pale yellow supernatant portion
(serum) of centrifuged blood sample or a urine sample, and induces reactions using reagents to
measure various components, such as sugar, cholesterol, protein, enzyme, etc.
 Why choose biochemistry analyzers –
• Significantly reduces repetitive tasks
• Handles loading, tube cleaning, mechanical control, and data processing
• Easy to operate (easy insertion of samples and automated programs)
• Speed up complex analysis
• Helps to streamlining daily laboratory activities
• To enable early detection as well as diagnosis of disease
• Comply with safety standards of the healthcare industry
• Provides a report as soon as possible (means, it provides the best quality of diagnostic
information).
Technique for the measurement process
 The figure above shows the outline of BioMajesty™
 The samples for BioMajesty™ are the pale yellow liquid
portion of blood called blood serum, and urine.
 A certain precise volume of sample (normally 30 micro liters) is
collected by using a dilution pipette, and diluted by 5 times
using a dilution disc.
 The measurable sample is 150 micro liters (μl). From this
dilution sample, a precisely-measured amount of 2 to 25
microliters per test item is transferred to a reaction cell (1 eye
drop in the rotating reactor is about 40 microliters).
 The reaction cell already contains reagent transferred from the
reagent bottle in reagent turntable 1.
 Reactions are induced in the samples in the reaction cells at a
temperature of 37μ. If a color reaction does not occur with one
kind of reagent, it is possible to add a second or third kind of
reagent (reagent turn table 2).
 After allowing the reaction to proceed for a certain time
(normally 10 minutes), the color density is measured by using a
colorimeter (in the figure, “multi-wavelength photometer”). The
mechanism of the colorimeter is to shine a light through the
sample being measured, and then electrically detect the amount
 The measured data is indicated numerically using A/D converters (analogue→digital converter),
calculated by CPU, and the results are output. BioMajesty™ offers high dispensing performance of
sample and reagent, reduction of residual water to the minimum, due to a cleaning mechanism for the
outside of the pipettes and measures to eliminate residual-water in the reaction cells, and acquisition
of good accuracy measurement data.
BLOOD CULTURE EQUIPMENT
 BLOOD CULTURE EQUIPMENT
 A blood culture is a laboratory test in which blood, taken from the patient, is inoculated into bottles
containing culture media to determine whether infection-causing microorganisms (bacteria or fungi) are
present in the patient’s bloodstream.
 Blood cultures are intended to:
• Confirm the presence of microorganisms
• in the bloodstream
• Identify the microbial etiology of
• the bloodstream infection
• Help determine the source of
• infection (e.g., endocarditis)
• Provide an organism for
• susceptibility testing and optimization
• of antimicrobial therapy
HOW A BLOOD CULTURE IS PERFORMED
 The blood draw may be performed in a hospital, emergency department, or specialized testing facility.
Blood cultures are rarely done in an outpatient setting.
 To start, your skin is cleaned to prevent any microorganisms on your skin from contaminating the test.
Your nurse or technician then usually wraps a cuff or an elastic band around your arm to allow your
veins to fill with blood and become more visible. They next use one needle to draw several samples of
blood from your arm.
 Multiple blood samples are generally collected from different veins to help increase the chance of
detecting the bacteria or fungi in your bloodstream. If you’re an adult, your doctor or healthcare team
usually collects two to three blood samples, often drawn on different visits.
 After the draw, your nurse or technician covers the puncture site with some gauze and a bandage. The
blood sample is then submitted to a laboratory where it’s cultured: Each blood sample is added to a
bottle containing a liquid known as broth. The broth encourages any microorganisms present in the blood
sample to grow.
PRINCIPLE OF BD BACTEC
 When microorganisms are present in culture vials, they
metabolize nutrients in the culture medium, releasing carbon
dioxide into the medium. A dye in the sensor at the bottom of the
vial reacts with CO2. This modulates the amount of light that is
absorbed by a fluorescent material in the sensor. A photodetector
at each station measures the level of fluorescence, which
corresponds to the amount of CO2 released by organisms. Then
the measurement is interpreted by the system according to pre-
programmed positivity parameters. At system startup, the
onboard computer performs self-diagnostics and downloads
operating instructions to the drawer rows. Then the instrument(s)
automatically begin testing. Light Emitting Diodes (LEDs)
behind the vials illuminate the rows, activating the vials’
fluorescent sensors. After a warm-up period, the instrument’s
photodetectors then take the readings. A test cycle of all rows is
completed every ten minutes. Positive cultures are immediately
flagged by an indicator light on the front of the instrument, an
audible alarm, and are displayed on the LCD display. When
BD BACTEC
positive vials are identified, the lab technologist pulls them from
the instrument for confirmation of results, and for isolation and
identification of the organism.
RIA (RADIO IMMUNOASSAY)
TECHNIQUE
 RIA (RADIO IMMUNOASSAY) TECHNIQUE
 RIA is an immunoassay technique developed by Berson and Yalow in 1960 to determine the
concentration of antigens (hormones, vitamins, serum protein, drugs etc) present in a sample
solution.
 Highly sensitive method which can detect .001ug of antigen in 1mL of sample.

In order to measure the concentration of A-antigen

in a serum sample, we require
• Anti-A Antibodies: binds to the A-antigen in the
sample and are prepared by hybridoma
technology.
• Radiolabelled A-Antigen: some part of this
antigen is prepared by radio isotopes which can
emit radiations like gamma or Beta rays. Heavy
isotopes of hydrogen (tritium) or Iodine-125 is
used for this.
• Unlabelled A Antigen: Same A-antigen in
unlabelled or original form.
 RIA METHODOLOGY
The test is performed by the following steps
 At first, the microtiter plate is coated with Anti-A Monoclonal antibodies.
 Radiolabel antigens are added to the well in an excess quantity.
 Since the concentration of the antigens are high, all the antigen binding sites of antibodies are
saturated by antigens and no antibodies remain free.
 Some radio labelled antigens remain free and the unbound antigens are removed by washing. The
radioactivity of antibodies is 100%.
 A known small amount of unlabeled antigen (1ng) is introduced into the well. This will result in a
competition between the radioactive antigen and the unlabelled antigen for binding antibodies. This
competition is proportional to the concentration of unlabeled antigen to the well. The competition is
less since we added only 1ng of unlabeled antigen.
 Few radio labelled antigens will be removed from the antibodies
and their place will be taken by the unlabeled antigens.
 The well is washed to remove the unbound antigens. The number of
radioactive antigens has decreased due to the addition of unlabeled
antigens and the radioactivity of the antibody is not 100%.
 The radioactivity reduces with the increased addition of unlabeled
antigens.
 Now we need to determine the A-Antigen in the serum sample. For
this, we have to repeat all the steps till the addition of radio labelled
antigens and instead of adding the unlabeled antigens, we need to
add the serum. The sample contains A-Antigen of unknown
concentration.
 The A-Antigen will compete with the radio labelled antigens and
remove them from antibodies. By measuring the percentage of
radioactivity, we can extrapolate this value on the standard graph to
obtain the concentration of A-Antigen
ADVANTAGES:
• Detection of hormones and allergies.

DISADVANTAGE:
• Hazardous to the researcher due to the usage of radio isotopes
• Expensive instrumentation
• Radio isotopes have short shelf life
• Time consuming technique
 ELISA (ENZYME LINKED
IMMUNOSORBENT ASSAY)
 ELISA (ENZYME LINKED
IMMUNOSORBENT ASSAY)
 The enzyme linked immunosorbent assay (ELISA) is a common laboratory technique used to measure the
concentration of an analyte (usually antibodies or antigens) in the body fluids to identify the type of
infection.
 Enzyme activity is measured using colorimetry principle.
 ELISA is also known as solid phase enzyme immune assay since it is performed on a solid phase
depending mainly on enzymes
 It is used to detect and quantify the protein in a protein mixture
 It is a labelled immunoassay as the antigen and antibody are labelled with the reporter enzyme
 Detects antibodies in patient specimen
Examples:
• Home pregnancy tests
• HIV tests
• Tests for some coagulation factors, cytokines, and autoantibodies
 METHODOLOGY OF ELISA
1. Add patient specimen to well coated with ligand
2. Add AHG with enzyme attached
3. Add substrate
4. Measure colour change
5. Colour change = patient has the antibody

THREE TYPES OF ELISA TECHNIQUE ARE:

 DIRECT ELISA TECHNIQUE
 IN-DIRECT ELISA TECHNIQUE
 SANDWICH ELISA TECHNIQUE
 DIRECT ELISA TECHNIQUE

1. The sample consist of different proteins and all these proteins are represented by different colours and
shapes.
2. For example, here we want to measure the green proteins in each sample and plot the graph of green
protein concentration in each sample.
3. The ELISA test is performed on a 96 wells polystyrene microtiter plate with straight bottom wells to stick
the proteins strongly to its surface.
4. When we add the samples to one of the wells, the proteins will stick to the surfaces of the plate after a
few minutes.
5. Now we add the specific detection antibody with its attached enzyme to the sample well and over time the
antibody gets attached to that specific proteins of its interest.
6. The remaining unbound antibodies are washed away.
7. Before adding the detection antibody, we need to add non reacting surface blocking proteins (BSA) to
avoid detection antibodies sticking to the surface resulting in wrong measurement.
8. Now to the sample, if we add a clear chemical, a blue colour change occurs due to its reaction with the
enzymes and again by adding some acid, the colour changes to yellow.
9. The enzyme produces a colour change which is used to measure the proteins.-
 INDIRECT ELISA TECHNIQUE
 In this method, we introduce two antibodies to the sample well.
 The first antibody which binds to the protein is the primary detection antibody
 The second antibody with the enzyme attached to it is the secondary detection antibody.
 The primary antibody is prepared by Injecting the protein of our interest in an animal (mouse, monkey etc)
and let the animal’s immune system produce the antibody specific for that protein and extract it.
 All the other process remains the same except the secondary detection antibody sticks to the its primary
detection antibody which in turn sticks to the protein of interest. The primary antibody made out from
some animal and the secondary antibody is species specific.

Advantages:
• More sensitive since the primary antibody can stick
to several secondary antibodies giving more
accurate results
• Less expensive than direct method.
 SANDWICH ELISA TECHNIQUE
1. In this method, we introduce a capture antibody before we add the sample to the well.
2. The capture antibody sticks specifically to the protein of interest.
3. The capture antibody is the first thing to stick to the surface and the remaining unsticked antibodies are
washed away.
4. Then the empty spaces are filled by the blocking proteins.
5. Now when we add the sample, the proteins of our interest will only stick to the antibodies and the
remaing proteins are washed away.
6. The detection antibodies are then introduced after which we add the chemical and acid which produces
the colour changes for measurement like direct ELISA method.

This method gives a more accurate result

since only the protein of our interest sticks
to the surface and has more sensitivity
compared to the other two methods
 APPLICATIONS OF ELISA

1. The presence of antibodies and antigens in a sample can be determined.

2. It is used in the food industry to detect any food allergens present.

3. To determine the concentration of serum antibody in a virus test.

4. During a disease outbreak, to evaluate the spread of the disease, e.g. during recent COVID-19
outbreak, rapid testing kits are being used to determine presence of antibodies in the blood sample.
CHEMILUMINESCENCE
 CHEMILUMINESCENCE
Luminiscence Process:
 Technique in which cold light can be emitted at low
temperature
 The source kicks an electron of an atom out of its lowest
energy ground state into a higher energy excited state.
 Electron returns to the ground state by emitting energy in the
form of light.
Chemiluminescence
 Light emission due to a chemical reaction. Similar to
fluorescence, there are some molecules that, due to their
structure, can produce light rather than heat when they react
with other molecules.
 Some immunological methods for measuring hormones and
tumour markers utilize a chemiluminescent molecule.
• After a selective antibody binds to the analyte in the patient’s specimen, the bound and unbound antibodies are
separated, and a second antibody linked to a chemiluminescent molecule is added.
• Once bound to the first antibody bound complex, a chemical is added to the mixture to generate a
chemiluminescent signal that is proportional to the amount of analyte in the sample.
• In a variation of this technique, the chemiluminescent signal is generated by pulsing the mixture with an
electrical current rather than through a chemical reaction. This method is called electrochemiluminescence
 CHEMILUMINESCENCE
 Unlike fluorescence spectroscopy, chemiluminescence’s energy
necessary to excite the analytes to higher electronic, vibrational, and
rotational states (from which they can decay be emission) does not come
from an external light source like a laser or lamp, the problem of
excitation source scattering is completely avoided.
 They both give off a photon as an electron relaxes from a higher energy
state to a lower energy state, but the difference lies in the method used
to excite that electron to a higher energy state in the first place.
 In fluorescence the electron is kicked up to a higher energy state by the
addition of a photon. In chemiluminescence the electron is in a high-
energy state due to the creation of an unstable intermediate in a
chemical reaction.
 Light is released when the intermediate breaks down into the final
products of the reaction.
Process
Process includes emission of light with limited emission of heat as the result of a chemical reaction.
If [A] + [B] [◊] [products]+ Light
[A], [B]: Reactants
[◊]: Excited Intermediates
For eg: 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 a 3-aminophthalate
3-APA [◊] is the excited state producing light as it decays to a lower energy level
CHEMILUMINESCENCE IMMUNOASSAY
ANALYZER (CLIA)
• Chemiluminescence immunoassays are used to estimate the analytes which have extremely low concentration in
the blood such as hormones, serological markers, drugs etc.
• It is a method to determine the concentration of samples according to the intensity of the luminescence that a
chemical reaction emits.
• High sensitivity, high throughput and economic way to quantitatively measure antigen in cell lysates, plasma,
urine, saliva, tissue and culture media samples

Applications:
• Thyroid function markers
• Tumour markers
• Diabetic marker
• Cardiac marker
 BASIC PRINCIPLE OF CLIA
• A Chemiluminescent acridinium labelled conjugate is added which binds the immune complex to complete the reaction mixture
• After a period of time, the reaction mixture is again washed to remove the unbound materials.

• The reaction mixture incubates and analyte present in the sample binds to the corresponding capture molecules on the micro particles
forming the immunocomplex.
• A Magnet attracts the paramagnetic particles bound to the specific micro particle analyte to the wall of the reaction vessel.
• Washing is done to remove all the unbounded materials.

• A Chemiluminescent acridinium labelled conjugate is added which binds the immune complex to complete the reaction mixture
• After a period of time, the reaction mixture is again washed to remove the unbound materials.
 CLIA
Add pre trigger which creates an acidic environment to prevent early release of energy or light emission.

• Trigger solution is now added to complete the

reaction.
• The resulting chemiluminescent reaction is measured
as relative light unit (RLU).

• A direct relationship exits between the amount of antigen or antibody in the sample and the relative light units
detected by architect system optics.
• The generated signal is directly proportional to the concentration of analyte.
IMMUNOPRECIPITATION ( IP)
 IMMUNOPRECIPITATION ( IP)
 IP is a technique that is based on the principle of antibody-antigen interaction to enrich or isolate a protein
from biological samples to study its identity, structure, expression and post-translational modifications.
Variations of IP are used to study the interaction between the target protein (known as a bait) with other
proteins (via Co-IP) or nucleic acids (via Chromatin IP or RNA-IP).
 In IP, an antibody is added first to a mixture containing an antigen, and incubated to allow antigen-
antibody complexes to form.
 Subsequently, the antigen-antibody complexes are incubated with an immobilized antibody against the
primary antibody (secondary antibody) or with protein A/G-coated beads to allow them to absorb the
complexes.
 The beads are then thoroughly washed, and the antigen is eluted from the beads by an acidic solution or
SDS.
 If suitable antibody is not available, the target molecule is fused to a His tag or other tags by recombinant
DNA techniques, and immunoprecipitated using an antibody to the tag (pull-down assay)
IMMUNO FLOURESCENCE (IF)
 IMMUNO FLOURESCENCE (IF)
 Immunofluorescence (IF) is an important immunochemical technique that allows
detection and localization of a wide variety of antigens in different types of tissues of
various cell preparations.

 These labeled antibodies bind directly or indirectly to cellular antigens. The

technique has a number of different biological applications including evaluation of
cells in suspension, cultured cells, tissue, beads and in microarrays.

 The fluorescent dye is subjected to short-wavelength, high energy light, which is

absorbed and emitted as light of a different wavelength. The emitted fluorescence has
a lower energy than the absorbed light, so the wavelength of the emitted light is
longer than that of the excitation light.

 The fluorescence can be visualized using fluorescence microscopy. The IF technique

allows for a visualization of the presence as well as the distribution of target
molecules in a sample.
 TYPES OF IMMUNOFLUORESCENCE
There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect).

PRIMARY (DIRECT)
 Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to a
fluorophore.

 The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be
detected via microscopy. This technique has several advantages over the secondary (or indirect)
protocol below because of the direct conjugation of the antibody to the fluorophore.

 This reduces the number of steps in the staining procedure making the process faster and can reduce
background signal by avoiding some issues with antibody cross-reactivity or non-specificity.

 However, since the number of fluorescent molecules that can be bound to the primary antibody is
limited, direct immunofluorescence is less sensitive than indirect immunofluorescence.
SECONDARY (INDIRECT)
 Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first (primary) antibody
specifically binds the target molecule, and the secondary antibody, which carries the fluorophore,
recognizes the primary antibody and binds to it.
 Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by
increasing the number of fluorophore molecules per antigen.
 This protocol is more complex and time consuming than the primary (or direct) protocol above, but it
allows more flexibility because a variety of different secondary antibodies and detection techniques can be
used for a given primary antibody.
POLYMERASE CHAIN REACTION
(PCR)
 POLYMERASE CHAIN REACTION (PCR)
 Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences. The method
involves using short DNA sequences called primers to select the portion of the genome to be amplified.
The temperature of the sample is repeatedly raised and lowered to help a DNA replication enzyme copy
the target DNA sequence. The technique can produce a billion copies of the target sequence in just a few
hours.
TAQ POLYMERASE
 Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of
DNA, using existing strands as templates.
 The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from
which it was isolated (Thermus aquaticus).
 T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most
active around 70 °\text C70°C70, °, start text, C, end text (a temperature at which a human or E. coli DNA
polymerase would be nonfunctional).
 This heat-stability makes Taq polymerase ideal for PCR. As we'll see, high temperature is used repeatedly in
PCR to denature the template DNA, or separate.

PCR PRIMERS
 Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a short sequence of
nucleotides that provides a starting point for DNA synthesis. In a PCR reaction, the experimenter determines
the region of DNA that will be copied, or amplified, by the primers she or he chooses.
 PCR primers are short pieces of single-stranded DNA, usually around 202020 nucleotides in length. Two
primers are used in each PCR reaction, and they are designed so that they flank the target region (region that
should be copied).
 That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at
the edges of the region to be copied. The primers bind to the template by complementary base pairing.
When the primers are bound to the template, they can be extended by the polymerase, and the region that lies between
them will get copied.
 THE STEPS OF PCR
 The key ingredients of a PCR reaction are Taq polymerase,
primers, template DNA, and nucleotides (DNA building
blocks). The ingredients are assembled in a tube, along with
cofactors needed by the enzyme, and are put through
repeated cycles of heating and cooling that allow DNA to be
synthesized.
 The basic steps are:
1. Denaturation (96 °\text C96°C96, °, start text, C, end text):
Heat the reaction strongly to separate, or denature, the DNA
strands. This provides single-stranded template for the next
step.
2. Annealing (555555 - 656565°\text C°C°, start text, C, end
text): Cool the reaction so the primers can bind to their
complementary sequences on the single-stranded template
DNA.
3. Extension (72 °\text C72°C72, °, start text, C, end text):
Raise the reaction temperatures so Taq polymerase extends
the primers, synthesizing new strands of DNA
 This cycle repeats 252525 - 353535 times in a typical PCR reaction, which generally takes 222 - 444 hours,
depending on the length of the DNA region being copied. If the reaction is efficient (works well), the target
region can go from just one or a few copies to billions.
 That’s because it’s not just the original DNA that’s used as a template each time. Instead, the new DNA
that’s made in one round can serve as a template in the next round of DNA synthesis. There are many
copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the number
of DNA molecules can roughly double in each round of cycling. This pattern of exponential growth is
shown in the image below.
RT-PCR INSTRUMENTATION
 RT-PCR
WHAT IS REAL TIME RT–PCR?
 It is a technique used to monitor the progress of a PCR reaction in real-time.
 At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified.
 It is based on the detection of the fluorescence produced by a reporter molecule which increases, as the
reaction proceeds.
 It is also known as a quantitative polymerase chain reaction (qPCR), which is a laboratory technique of
molecular biology based on the polymerase chain reaction (PCR).
 qPCR is a powerful technique that allows exponential amplification of DNA sequences.
 A PCR reaction needs a pair of primers that are complementary to the sequence of interest. Primers are
extended by the DNA polymerase.
 The copies produced after the extension, so-called amplicons, are re-amplified with the same primers
leading thus to exponential amplification of the DNA molecules.
 After amplification, however, gel electrophoresis is used to analyze the amplified PCR products and this
makes conventional PCR time consuming; since the reaction must finish before proceeding with the post-
PCR analysis. Real-Time PCR overcomes this problem.
 The term “real-time” denotes that it can monitor the progress of the amplification when the process is going
on in contrast to the conventional PCR method where analysis is possible only after the process is
completed.
PRINCIPLE OF REAL TIME PCR
 This same principle of amplification of PCR is employed in real-time PCR. But instead of looking at bands
on a gel at the end of the reaction, the process is monitored in “real-time”. The reaction is placed into a real-
time PCR machine that watches the reaction occur with a camera or detector.
 Although many different techniques are used to monitor the progress of a PCR reaction, all have one thing in
common. They all link the amplification of DNA to the generation of fluorescence which can simply be
detected with a camera during each PCR cycle. Hence, as the number of gene copies increases during the
reaction, so does the fluorescence, indicating the progress of the reaction.
A. AMPLIFICATION
Denaturation
 High temperature incubation is used to “melt” double- stranded DNA into single strands and loosen secondary structure
in single-stranded DNA. The highest temperature that the DNA polymerase can withstand is typically used (usually
95°C). The denaturation time can be increased if template GC content is high.
Annealing
 During annealing, complementary sequences have an opportunity to hybridize, so an appropriate temperature is used that
is based on the calculated melting temperature (Tm) of the primers(5°C below the Tm of the primer).
Extension
 At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per
second. When an amplicon in real-time PCR is small, this step is often combined with the annealing step using 60°C as
the temperature.
B. DETECTION
 The detection is based on fluorescence technology.
 The specimen is first kept in proper well and subjected to thermal cycle like in the normal PCR.
 The machine, however, in the Real Time PCR is subjected to tungsten or halogen source that lead to fluoresce the marker
added to the sample and the signal is amplified with the amplification of copy number of sample DNA.
 The emitted signal is detected by an detector and sent to computer after conversion into digital signal that is displayed on
screen.
 The signal can be detected when it comes up the threshold level (lowest detection level of the detector).
ELECTROPHORESIS
 ELECTROPHORESIS
 Electrophoresis is the essential physical laboratory technique of analysis of nucleic acids. It involves the
compounds which can acquire electric charge in conducting electrodes. It is basically the movement of
charged particles under the influence of an external electric field.
 This technique is used for the separation and analysis of nucleic acids i.e., DNA, RNA, and for proteins. The
separation is based on the size, charge, density, and purity of molecules.

PRINCIPLE OF ELECTROPHORESIS
 Electrophoresis is defined as the migration of charged particles, under the influence of an electric field at a
definite pH.
 In a mixture of proteins, each protein with its electrical charge will move differently in an electric field.
 This electrophoretic mobility depends on the pH of the medium, strength of the field, net charge of the
molecule and size/shape of the molecule.
 Electrophoresis is used for the analysis of large molecules (proteins and nucleic acids) and simpler charged
molecules (peptide, simpler ions).
APPLICATIONS OF ELECTROPHORESIS
 Sizing of nucleic acid molecules
 DNA fragmentation for southern blotting
 RNA fragmentation for northern blotting
 Protein fragmentation for western blotting
 Analytical separation of PCR products
 Detection and analysis of mutations or variations in sequences.

TYPES OF ELECTROPHORESIS
 Paper electrophoresis
 Zone Electrophoresis
 Continuous Electrophoresis
 Agarose gel electrophoresis
 Polyacrylamide (PAGE)
 Pulsed Field Electrophoresis
 Capillary Electrophoresis
 Routine Electrophoresis
CHROMATOGRAPHY
 CHROMATOGRAPHY
 Chromatography is an important biophysical technique that enables the separation, identification, and
purification of the components of a mixture for qualitative and quantitative analysis.
CHROMATOGRAPHY PRINCIPLE
 Chromatography is based on the principle where molecules in mixture applied onto the surface or into the
solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a
mobile phase.
 The factors effective on this separation process include molecular characteristics related to adsorption
(liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights.
 Because of these differences, some components of the mixture stay longer in the stationary phase, and they
move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the
system faster.
 Based on this approach three components form the basis of the chromatography technique.
• Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the
surface a solid support”.
• Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
• Separated molecules
 Chromatography methods based on partition are very effective on separation, and identification of
small molecules as amino acids, carbohydrates, and fatty acids.
 However, affinity chromatographies (ie. ion-exchange chromatography) are more effective in the
separation of macromolecules as nucleic acids, and proteins.
GAS CHROMATOGRAPHY
 GAS CHROMATOGRAPHY
GAS CHROMATOGRAPHY
 Chromatography is a technique that separates components in a mixture by the difference in
partitioning behavior between mobile and stationary phases. Gas chromatography (GC) is one of the
popular chromatography techniques to separate volatile compounds or substances. The mobile phase
is a gas such as helium, and the stationary phase is a high-boiling liquid that is adsorbed on a solid.
Because of its simplicity, high sensitivity, and the ability to effectively separate mixtures, gas
chromatography has become one of the most important tools in chemistry.
THE PRINCIPLE OF GAS CHROMATOGRAPHY
 Components in the mixture are distributed between two phases, one of which is a stationary phase,
and the other is a mobile phase gas, or carrier gas, that carries the mixture through the stationary
phase. Compounds in the mobile phase interact with the stationary phase as they pass through. Due to
the differences in properties and structures of each component, the size and affinity of each
interaction with the stationary phase are different. Therefore, under the same driving force, the
retention time of different components differs in the column, thus moving out of the column in
different orders.
Applications
 GC analysis is used to calculate the content of a
chemical product, for example in assuring the quality
of products in the chemical industry; or measuring
toxic substances in soil, air or water.
 Gas chromatography is used in the analysis of:
(a) air-borne pollutants
(b) performance-enhancing drugs in athlete’s urine
samples
(c) oil spills
(d) essential oils in perfume preparation
 GC is very accurate if used properly and can
measure picomoles of a substance in a 1 ml liquid
sample, or parts-per-billion concentrations in gaseous
samples.
 Gas Chromatography is used extensively in forensic
science. Disciplines as diverse as solid drug dose (pre-
consumption form) identification and quantification,
arson investigation, paint chip analysis, and toxicology
cases, employ GC to identify and quantify various
biological specimens and crime-scene evidence.
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 High performance liquid chromatography or commonly known as HPLC is an analytical technique used to
separate, identify or quantify each component in a mixture.
 The mixture is separated using the basic principle of column chromatography and then identified and
quantified by spectroscopy.
 In the 1960s the column chromatography LC with its low-pressure suitable glass columns was further
developed to the HPLC with its high-pressure adapted metal columns.
 HPLC is thus basically a highly improved form of column liquid chromatography. Instead of a solvent being
allowed to drip through a column under gravity, it is forced through under high pressures of up to 400
atmospheres.

PRINCIPLE OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

 The purification takes place in a separation column between a stationary and a mobile phase.
 The stationary phase is a granular material with very small porous particles in a separation column.
 The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at high pressure through
the separation column.
 Via a valve with a connected sample loop, i.e. a small tube or a capillary made of stainless steel, the
sample is injected into the mobile phase flow from the pump to the separation column using a
syringe.
 Subsequently, the individual components of the sample migrate through the column at different rates
because they are retained to a varying degree by interactions with the stationary phase.
 After leaving the column, the individual substances are detected by a suitable detector and passed on
as a signal to the HPLC software on the computer.
 At the end of this operation/run, a chromatogram in the HPLC software on the computer is obtained.
 The chromatogram allows the identification and quantification of the different substances.
INSTRUMENTATION OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
The Pump
• The development of HPLC led to the development of the pump system.
• The pump is positioned in the most upper stream of the liquid chromatography system and generates a flow
of eluent from the solvent reservoir into the system.
• High-pressure generation is a “standard” requirement of pumps besides which, it should also to be able to
provide a consistent pressure at any condition and a controllable and reproducible flow rate.
• Most pumps used in current LC systems generate the flow by back-and-forth motion of a motor-driven
piston (reciprocating pumps). Because of this piston motion, it produces “pulses”.
Injector
• An injector is placed next to the pump.
• The simplest method is to use a syringe, and the sample is introduced to the flow of eluent.
• The most widely used injection method is based on sampling loops.
• The use of the autosampler (auto-injector) system is also widely used that allows repeated injections in a set
scheduled-timing.
Column
• The separation is performed inside the column.
• The recent columns are often prepared in a stainless steel housing, instead of glass columns.
• The packing material generally used is silica or polymer gels compared to calcium carbonate.
The eluent used for LC varies from acidic to basic solvents.
• Most column housing is made of stainless steel since stainless is tolerant towards a large variety of solvents.
Detector
• Separation of analytes is performed inside the column, whereas a detector is used to observe the obtained
separation.
• The composition of the eluent is consistent when no analyte is present. While the presence of analyte changes
the composition of the eluent. What detector does is to measure these differences.
• This difference is monitored as a form of an electronic signal. There are different types of detectors available.
Recorder
• The change in eluent detected by a detector is in the form of an electronic signal, and thus it is still not visible to our
eyes.
• In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a computer-based data processor
(integrator) is more common.
• There are various types of data processors; from a simple system consisting of the in-built printer and word
processor while those with software that are specifically designed for an LC system which not only data acquisition
but features like peak-fitting, baseline correction, automatic concentration calculation, molecular weight
determination, etc.
Degasser
• The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our eyes.
• When gas is present in the eluent, this is detected as noise and causes an unstable baseline.
• Degasser uses special polymer membrane tubing to remove gases.
• The numerous very small pores on the surface of the polymer tube allow the air to go through while preventing any
liquid to go through the pore.
Column Heater
• The LC separation is often largely influenced by the column temperature.
• In order to obtain repeatable results, it is important to keep consistent temperature conditions.
• Also for some analysis, such as sugar and organic acid, better resolutions can be obtained at elevated temperatures
(50 to 80°C).
Applications of High-Performance Liquid Chromatography (HPLC)
The HPLC has developed into a universally applicable method so that it finds its use in almost all areas of
chemistry, biochemistry, and pharmacy.

• Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics
• Determination of drugs in biological matrices
• Isolation of valuable products
• Product purity and quality control of industrial products and fine chemicals
• Separation and purification of biopolymers such as enzymes or nucleic acids
• Water purification
• Pre-concentration of trace components
• Ligand-exchange chromatography
• Ion-exchange chromatography of proteins
• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides
PAPER CHROMATOGRAPHY
 PAPER CHROMATOGRAPHY
What Is Paper Chromatography?
Chromatography technique that uses paper sheets or strips as the adsorbent being the stationary
phase through which a solution is made to pass is called paper chromatography. It is an inexpensive
method of separating dissolved chemical substances by their different migration rates across the
sheets of paper. It is a powerful analytical tool that uses very small quantities of material. Paper
chromatography was discovered by Synge and Martin in the year 1943.

Paper Chromatography Principle

The principle involved can be partition chromatography or adsorption chromatography. Partition
chromatography because the substances are partitioned or distributed between liquid phases. The two
phases are water held in pores of the filter paper and the other phase is a mobile phase which passes
through the paper. When the mobile phase moves, the separation of the mixture takes place. The
compounds in the mixture separate themselves based on the differences in their affinity towards
stationary and mobile phase solvents under the capillary action of pores in the paper. Adsorption
chromatography between solid and liquid phases, wherein the solid surface of the paper is the
stationary phase and the liquid phase is the mobile phase.
Paper Chromatography Procedure
Below we have explained the procedure to conduct Paper Chromatography Experiment for easy understanding of
students.
1. Selecting a suitable type of development: It is decided based on the complexity of the solvent, paper,
mixture, etc. Usually ascending type or radial paper chromatography is used as they are easy to perform. Also,
it is easy to handle, the chromatogram obtained is faster and the process is less time-consuming.
2. Selecting a suitable filter paper: Selection of filter paper is done based on the size of the pores and the
sample quality.
3. Prepare the sample: Sample preparation includes the dissolution of the sample in a suitable solvent (inert
with the sample under analysis) used in making the mobile phase.
4. Spot the sample on the paper: Samples should be spotted at a proper position on the paper by using a
capillary tube.
5. Chromatogram development: Chromatogram development is spotted by immersing the paper in the mobile
phase. Due to the capillary action of paper, the mobile phase moves over the sample on the paper.
6. Paper drying and compound detection: Once the chromatogram is developed, the paper is dried using an air
drier. Also, detecting solution can be sprayed on the chromatogram developed paper and dried to identify
the sample chromatogram spots.
PAPER CHROMATOGRAPHY APPLICATIONS
There are various applications of paper chromatography. Some of the uses of Paper Chromatography in
different fields are discussed below:
• To study the process of fermentation and ripening.
• To check the purity of pharmaceuticals.
• To inspect cosmetics.
• To detect the adulterants.
• To detect the contaminants in drinks and foods.
• To examine the reaction mixtures in biochemical laboratories.
• To determine dopes and drugs in humans and animals.
FLOWCYTOMETRY
FLOWCYTOMETRY
Flowcytometry principle: the passage of cell
passed through an orifice, additionally a laser is
directed at them, and the scattered light is
measured at different angles, as well as cells are
counted and sorted.

• Cell components are fluorescently labelled

and then excited by the laser to emit light at varying
wavelengths.
• Cells pass through sheath fluid, one cell at
time, by hydro-dynamic focusing the laser light is
directed towards the stream which carries the cell.
Once the light is focused on the cell the light is scattered
in multiple angles:
FSC (0^0) – information related to cell
volume.
SSC ( 〖 90 〗 ^0)- information related to
cells internal complexity.
Forward high-angle scatter (5- 〖 15 〗 ^0)-
cell’s complexity.
Forward low-angle scatter (2-3^0)- cell’s
volume
Collection optics consists of collection of lenses to collect light emitted from
particle-laser beam interaction and a system of optical mirrors and filters to separate
and then direct specified wavelength of the collected lights to the appropriate
detectors.
Using detectors, we can particularly measure fluorescent dye by placing suitable
detectors, such as long pass, short pass and band pass filters.
• Band pass filters allows only narrow range of wavelength, which is close
to the emission peak of the fluorescent dye to reach the detector.
• Long pass filters transmit wavelength of light equal to or longer than the
specified wavelength.
• Short pass filters transmit wavelength of light equal to or shorter than the
specified wavelength.

Detection and conversion of scattered light is accomplished by

photodetectors at specific angles. 2 types of detectors:
Photodiodes and photomultipliers.
• Photodiodes (PD) detect stronger signal such as FSC.
• Photomultiplier tubes (PMT): the photocathode of
PMT has greater sensitivity, they are known convert
photoelectrons more efficiently, detect weaker signals such SSC
and Fluorescence.
APPLICATIONS FOR FLOW CYTOMETRY
There are a number of applications for flow cytometry, including, but not limited to:
Immunophenotyping- Using fluorescence-conjugated antibodies directed toward a protein(s) of interest,
cells expressing that protein(s) on the surface or intracellularly may be detected by flow cytometry.Specific
cell types may be distinguished within a mixed population using multiple fluorescence-conjugated
antibodies.
Transfection efficiency may be determined when a fluorescent protein (i.e. GFP) is used as a marker.
Apoptosis measurement- Several flow cytometric methods to detect apoptosis are available. Cells can be
stained with Annexin V or 7ADD.
Cell cycle analysis- Fixed cells are stained with a dye that binds to DNA (e.g. propidium iodide,ethidium
bromide,DAPI).
Fluorescent intensity is used to determine the amount of cellular DNA present in each cell (i.e. two copies
of a genome have roughly twice the fluorescent intensity of one copy).
Cell proliferation- Carboxyfluorescein diacetate, succinimidyl ester (CFSE) is a dye that diffuses into cells
and is passed from parent to daughter cells so that the cells of each generation have half the fluorescent
intensity of their parent cells.
Cell sorting- A particular subset(s) of cells may be sorted from a mixture of cells based upon particular
properties.
Membrane potential- Bacterial membrane potential may be analyzed using DiOC2, which exhibits green
fluorescence in all bacterial cells, but shifts to red fluorescence as the dye becomes more concentrated in
cells with larger membrane potentials.Mitochondrial membrane potential may be analyzed in the same
manner with JC-1.
Live/dead bacteria discrimination- You can test how fast an antibiotic is killing microbes: live cells have
intact membranes and are impermeable to dyes such as propidium iodide, which only leaks into cells with
compromised membranes. Thiazole orange enters all cells, live and dead, to varying degrees. Thus a
combination of these two dyes provides a rapid and reliable method for discriminating live and dead
bacteria.
BLOOD CELL COUNTERS
 BLOOD CELL COUNTERS
 Haematology analyser also called as cell counters, it is the study of blood in
relation to health and disease, helps in identifying and measuring cells that
are presented in the blood.
3-part differential cell counter: Lymphocytes, Mid-cell and granulocytes
 3-part differential working principle
• Electrical impedance method
• Photometry
• Radiofrequency

COULTER COUNTERS/ELECTRICAL IMPEDANCE METHOD

 All haematology analysers use Coulter’s Principle. A 3-part differential cell
counter uses Coulter’s Principle to determine the size and volume of the
cells. Coulter’s principle is applied using two electrodes. Using
hydrodynamic focusing, the sample cells are sent through an orifice one cell
at a time. As the cells go through the orifice, they briefly cause electrical
resistance to the current. This resistance is recorded, measured, amplified,
and processed which3-part differential cell-counter can then be interpreted
by the computer into a histogram.
BLOOD GAS ANALYSER
 BLOOD GAS ANALYSER
WHAT IS A BLOOD GAS TEST?
 A blood gas test measures the amount of oxygen and carbon dioxide in the blood. It may also be used to
determine the pH of the blood, or how acidic it is. The test is commonly known as a blood gas analysis or arterial
blood gas (ABG) test.
 Your red blood cells transport oxygen and carbon dioxide throughout your body. These are known as blood gases.
 As blood passes through your lungs, oxygen flows into the blood while carbon dioxide flows out of the blood
into the lungs. The blood gas test can determine how well your lungs are able to move oxygen into the blood and
remove carbon dioxide from the blood.
HOW IS A BLOOD GAS TEST PERFORMED?
 A blood gas test requires the collection of a small sample of blood. Arterial blood can be obtained from an artery
in your wrist, arm, or groin, or preexisting arterial line if you are currently hospitalized. A blood gas sample can
also be venous, from a vein or preexisting IV or capillary, which requires a small prick to the heel.
 A healthcare provider will first sterilize the injection site with an antiseptic. Once they find an artery, they’ll insert
a needle into the artery and draw blood. You might feel a slight prick when the needle goes in. Arteries have more
smooth muscle layers than veins, and some may find an arterial blood gas test more painful than a blood draw
from a vein.
 After the needle is removed, the technician will hold pressure for a few minutes before putting a bandage over the
puncture wound.
 The blood sample will then be analyzed by a portable machine or in an on-site laboratory. The sample must be
 ANK Y O U
TH