PSZOP109: LAB EXERCISES BASED ON PSZO108

(60 HRS)

(Note: This practical manual is for reference purpose. The teachers may alter or modify the
protocols of experiment as per to their requirements/availability of facility.
Copyright to Publisher
 INDEX
Sr. No. Title of the Experiment Pg. No.

To study the pH of water sample/ soil sample using universal indicator/

1 2
pH paper/ pH meter.
To study structure, working, use and care of microscopes (dissecting
2 5
microscope and compound microscope).
Study of zooplanktons and phytoplanktons from
3 river/pond/lake/estuaries water sample using compound microscope 12
(Temporary mounting).

4 Separation of lipids in a given sample by TLC. 17

Separation of pigments from leaves or flowers by adsorption column

5 19
chromatography.
Separation and identification of amino acids by 2D paper
6 21
chromatography.
Study of following instruments through photographs:
7 23
Spectrophotometer, Flame photometer, SEM, TEM, HPLC, GCMS.
Colorimetric estimation of serum/egg protein by Peterson‐Lowry
8 29
method.

9 Separation of proteins using AGE or PAGE. 31

Field visit to any instrumentation laboratory/research

10 38
institute/centralized laboratory
.

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 Practical 1
Aim: To study the pH of water sample/ soil sample using universal indicator/ pH paper/ pH
meter.

Background Information:
The term “pH” refers to the measurement of hydrogen ion activity in the solution. Since the direct
measurement of the pH is very difficult, specific electrodes are needed for quick and accurate pH
determination. pH is measured on a scale of 0 to 14, with lower values indicating high H+ (more acidic)
and higher values indicating low H+ ion activity (less acidic). A pH of 7 is considered as neutral. Every
whole unit in pH represents a ten-fold increase in or decrease in hydrogen ion concentration. Most
natural waters possess the pH values ranging from 5.0 to 8.5. Rain water have a pH value of 5.4 to 6.0
which then reacts with the soils and minerals causing the reduction in H+ ion concentration and thus
the water may become alkaline with a pH 0f 8.0-8.5. More acid water (pH9) and other immediate
changes in the hydrogen ion concentration (pH) suggest that the quality of the water is adversely
affected due to the introduction of some toxic contaminants in water bodies.

Soil may be acidic, neutral or alkaline. Soil pH is very important because certain plants will only grow
successfully in soils of a certain pH. For example, heathers will grow successfully in acidic soils whilst
carnations will grow well in alkaline soils. Most plants will grow well in neutral soils.

pH is measured using pH meter, which comprises a detecting unit consisting of a glass electrode,
reference electrode, usually a calomel electrode connected by KCl Bridge to the pH sensitive glass
electrode and an indicating unit which indicates the pH corresponding to the electromotive force is
then detected. Before measurement, pH meter should be calibrated by using at least two buffers.

Requirement:
pH paper, Universal Indicator, pH meter, pH electrode filled with KCL solution, buffer solutions of
pH4 and pH 7 4, barium sulphate, clean beakers, test-tube, test-tube stand, tissue papers, distilled
water, thermometer, water sample, soil sample.

Principle:
When the pair of electrodes or a combined electrode (glass electrode and calomel electrode) is dipped
in an aqueous solution, a potential is developed across the thin glass of the bulb (of glass electrode).
The e. m. f. of complete cell (E) formed by the linking of these two electrodes at a given soln. temp.
is therefore E = E ref – E glass. E ref is the potential of the stable calomel electrode which at normal
room temp. is +0.250V. E glass is the potential of the glass electrode which depends on the pH of the
soln. under test. The resultant e.m.f. can be recorded potentiometrically by using vacuum tube
amplifier. Variations of pH with E may be recorded directly on the potentiometer scale graduated to
read pH.

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Procedure:

Using Ph meter:
1. Plug in the pH meter to power source and let it warm up for 5 to 10 minutes.
2. Wash the glass electrode with distilled water and clean slowly with a soft tissue.
3. Note the temperature of water and set the same on the pH meter.
4. Place the electrode in pH 7 buffer solution and set the value of 7 on the pH meter turning the
Calibrate knob on the meter.
5. Take out the electrode, wash with DW and clean.
6. Dip the electrode in the pH 4 buffer solution. Adjust the value on the pH readout meter by the
Slope switch.
7. Repeat with pH 7 and pH4 buffers till a correct and stable reading is displaced.
8. While moving and cleaning the electrode, put the selector switch on standby mode. Turn to pH
mode for recording the pH.
9. Now place the electrode in the water sample whose pH is to be determined.
10. You can take a number of simultaneous readings for different samples until the power is on

Using pH paper:
1. Take six strips pH paper and place them on a glazed tile. Mark them 1 to 6.
2. Take the test solutions in separate test tubes. Dissolve the solid substance by adding distilled
water to it. Label the test tubes.
3. Now, place a drop of the test solution on one strip of the pH paper with the help of a fine
dropper or glass rod. Use a fresh dropper for each test solution.
4. Observe the colour produced and match it with the different colour shades of the standard
colour pH chart.
5. Note down the colour of the pH from the colour chart that matches most closely with the colour
produced on the pH paper.
6. Similarly, find the pH value of the remaining samples by using a fresh strip of pH paper and a
separate glass rod or fine dropper for each one.

Using universal indicator:

1. Use a spatula to put a small amount of barium sulphate into a dry test tube. It should be dry to
stop the powder from sticking to the side of the tube.
2. Add a small amount of the soil to be tested (again using a spatula).
3. Use distilled water to fill the test tube to around ¾ full.
4. Add 5-6 drops of Universal indicator.
5. Put a bung in the tube, shake it well and allow to stand for short time.
6. A clear coloured liquid will be left in the top of the tube. This can then be compared with the
pH chart to find the pH of the soil being tested.
Soil pH
<5 5.5 6 6.5-7.5 7.5-8.5 >8.5

Strongly Moderately Slightly Neutral Moderately Strongly

acidic acidic acidic Appropriate for alkaline alkaline
most crops.

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Observation table:

Sample Colour pH Nature

Result:

Conclusion:

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 Practical 2

Aim: To study structure, working, use and care of microscopes (dissecting microscope and
compound microscope).

The term microscope can be split into two separate words, ‘micro’ and ‘scope’, where the term ‘micro’
means small or tiny, and ‘scope’ means to view or to observe. Therefore, a microscope can be
understood as an instrument to observe tiny elements. The optical microscope often referred to as the
light microscope, is a type of microscope that uses visible light and a system of lenses to magnify
images of small subjects.
There are two basic types of optical microscopes:
1. Simple microscopes
2. Compound microscopes.

Dissecting Microscope:
Dissecting Microscope or Stereoscopic microscopes are another name for these. Dissection
microscope is a digital optical microscope having low magnification power (5x-250x) which uses light
reflected from the specimen’s surface instead of light reflected from the specimen itself. Its principal
function is to dissect specimens as well as observe as well as analyze the dissected samples
qualitatively.

Principle of Dissecting Microscope:

The working principle of a dissecting microscope is that when a sample is placed within the focus of
the microscope, a virtual, erect and magnified image is obtained at the least distance of distinct vision
from the eye that is held at the lens.

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Structure of Dissecting Microscope:

1. Foot or Base:
It is the basal, horse-shoe shaped or circular part of dissecting microscope. It is made of heavy material.
It provides support to other parts of microscope.
2. Stand:
It is short but strong, hollow cylindrical rod. It’s one end is fixed at the foot or base. It provides support
to the mirror, adjustment screw and other parts.
3. Vertical Limb:
It is short and movable rod that fits into the hollow tube of the stand. With the help of the adjustment
screw, this limb can be moved up and down.
4. Folder Arm:
It is a horizontal arm. It’s one end is attached with the vertical limb and on it’s another end is attached
lens. Folded arm is movable. It can be moved up and down as well as left and right.
5. Lens:
It is a simple convex lens of either 2X, 3X, 5X, 10X or 20X magnification.
6. Stage:
It is rectangular glass plate attached to the upper end of the stand or limb. Slide or the object, to be
observed, is kept on the stage.
7. Clips:
Two clips are fitted on the stage. They are used to hold the slide in the desired position.
8. Adjustment Screw:
This is a screw used to adjust or move the vertical limb up and down.
9. Mirror:
It is concave reflecting mirror attached to the lower inner side of the stand. Light rays are reflected or
focused on the stage by the mirror.

Working:
1. Place the slide or the object, to be observed, on the stage.
2. Bring the lens over the slide with the help of folded arm.
3. Put the clips on two ends of the slide to keep it in position.
4. Move the vertical limb up and down by adjustment screw to bring the slide into desired focus.
5. Adjust the light over the slide with the help of the mirror and observe.
6. In case some material is to be dissected, place the material over the slide, repeat the entire
process mentioned above, dissect the material with the help of needles or other instruments,
and observe.
Utility:
Dissecting microscope is used to dissect small organisms or organs, e.g., embryo dissection. Its special
utility is to observe such materials where high magnification is not needed.

Application of Dissecting Microscope:

• It is common among the watchmakers as they can view a magnified image of the smallest parts.
• It is also used by the jewelers for obtaining a magnified image of the fine parts of the jewelry.
• Most educational institutions such as schools and colleges use a simple microscope in their
laboratories.
• Dermatologists (skin specialists) use simple microscopes to identify different skin disease

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Compound Microscope:
The term “compound” in compound microscopes refers to the microscope having more than one lens.
Devised with a system of combination of lenses, a compound microscope consists of two optical parts,
namely the objective lens and the ocular lens.

Principle of Compound Microscope:

A compound microscope is considered to be one of the standard microscopes that can be used for
general purposes. The arrangement of the lens is such that it magnifies the objects from the complex
system. There are two types of lenses that are used in the compound microscope:
• The objective lens is placed close to the object that needs to be examined.
• The eyepiece allows the image to be viewed.

The eyepiece is also known as the ocular lens. The light is made to pass through the thin transparent
object. A magnified image of the object is obtained by the objective lens. This image is known as the
real image. The eyepiece or the ocular lens then magnifies the real image more and is viewed as the
virtual image. The compound microscope is also known as the bright-field microscope because the
light passes directly through the light source to the eye through the two lenses. This mechanism makes
the field of vision brightly illuminated.

Parts of Compound Light Microscope:

Eyepiece And Body Tube

• The eyepiece is the lens through which the viewer looks to see the specimen.
• It usually contains a 10X or 15X power lens.
• The body tube connects the eyepiece to the objective lenses.
Objectives and Stage Clips
•Objective Lenses are one of the most important parts of a Compound Microscope.
• They are the closest to the specimen.
• A standard Microscope has three to four Objective Lenses which range from 4X to 100X.
• Stage Clips are metal clips that held the slide in place.
Arm and Base
• The Arm connects the Body Tube to the base of the Microscope.
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• The Base supports the Microscope and its where Illuminator.
Illuminator and Stage
• The illuminator is the light source for a microscope.
• A compound light microscope mostly uses a low voltage bulb as an illuminator.
• The stage is the flat platform where the slide is placed.
Nosepiece and Aperture
• Nosepiece is a rotating turret that holds the objective lenses.
• The viewer spins the nosepiece to select different objective lenses.
• The aperture is the middle of the stage that allows light from the illuminator to reach the specimen.

Condenser, Iris diaphragm, and Diaphragm

• A condenser gathers and focuses light from the illuminator onto the specimen being viewed.
• Iris diaphragm adjusts the amount of light that reaches the specimen.
• The diaphragm is a five-holed disk placed under the stage.
• Each hole is of a different diameter. By turning it, you can vary the amount of light passing through
the stage opening.

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Working:
1. Carry the microscope with two hands, one under the base and the other gripping the arm or
frame.
2. Gently place the microscope on a flat, level surface and plug into a power source. Some
microscopes have a built-in light source but others have a mirror to focus natural light or an
external light source.
3. With a built-in light source, turn on the light source and adjust the light setting so that it is not
too bright by turning or sliding the brightness adjustment knob on the base.
4. ‘If using an external light source direct the light via the mirror. Rotate the low power objective
into position. Remove the eyepiece, look down the body tube and adjust the mirror and
diaphragm setting so light is reflected up the tube and a circle of evenly illuminated light is
visible in the field of view. Replace the eyepiece. Use the concave mirror side if the microscope
has a fixed condenser lens or the flat mirror side if the microscope has an adjustable condenser’
5. The iris diaphragm is located just above the light source on the bottom side of the stage. Using
the lever attached, you can increase or decrease the amount of light reaching the specimen.
Look through the eyepiece and adjust the sub-stage iris diaphragm to allow sufficient
comfortable light through.
6. Between the stage and the iris diaphragm is the condenser. The condenser further aids in the
focusing of the light onto the specimen. In some microscopes it can be moved up and down.
To begin with, position it close to the stage. If you have a problem focusing your specimen then
adjust the position of the condenser.
7. Adjust the stage down as low as possible with the coarse focus knob.
8. Begin by viewing the specimen with the lowest power objective lens in place and then increase
to the higher power objective lenses.
9. Select the 4x scanning objective by rotating the nosepiece, ensuring it clicks into place.
10. Place a prepared slide onto the stage and hold it in place with the metal clips. Centre it so that
the specimen is under the objective lens. Move it with the stage control knobs either left to
right or backwards and forwards.
11. After placing the slide on the stage look at the objective lens and the stage from the side and
use the coarse focusing knob to bring the slide as close to the objective as possible without
touching it. Look in the eyepiece/s and slowly move the stage away from the objective lens
with the coarse focusing knob. Stop when the image comes into view.
12. If using a binocular microscope adjust the distance between the eyepieces to suit your eyes by
sliding the eyepieces in and out until you see one image. This is called the interocular distance.
13. Use the fine focus to sharpen the image. Scan the slide, select the part of the specimen you are
interested in and center it in your field of view. Adjust the sub-stage iris diaphragm to optimize
the lighting.
14. Rotate in the low power 10x objective and refocus with the fine focus. You may need to open
the iris diaphragm to let lighter in. In general, the higher the power, the lighter you require.
15. Repeat with the high power 40x objective, adjusting the iris diaphragm if required. Use only
the fine adjustment knob to focus the microscope when using the higher power objective lenses.
16. If you have a 100x oil immersion objective, you will need to first focus on the specimen with
the 40x objective. Next rotate the nosepiece so that a midway position is obtained between the
40x objective and the 100x objective. Place a small drop of immersion oil onto the slide

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 coverslip then continue to rotate the nosepiece so that the 100x objective is rotated into the oil.
The immersion oil should be used sparingly. Never use immersion oil with any of the other
objectives. (N.B. It is possible to place the oil directly on a specimen that has been fixed or
heat fixed and stained without a coverslip, e.g. bacterial slides. However, it is difficult to
remove the oil from the slide without damaging the smear.) Any attempt to re-look at the slide
with a low or high-power objective may result in contamination of these objectives with the
immersion oil. Do not use immersion oil on a wet mount unless you can secure the coverslip
well.
17. Sharpen the image with the fine focus only and adjust the light with the iris diaphragm if
required. When finished, lower the stage, rotate the low power objective (4x) into position and
remove the slide.
18. Clean the oil off the slide and the objective when finished with lens tissue and lens cleaning
fluid. In order to return to work at the lower magnifications, the slide must be completely
cleaned of any residual oil. Wipe the stage clean with a paper towel.
19. Turn off the light and at the main switch.
20. Report any problems to your teacher.
21. Cover the microscope with its dust cover.

Maintenance:

Tip 1: Handle with care

Most microscope problems occur as a result of improper handling. When carrying your microscope,
hold it by the base and the metal support arm. Do not pick it up by the stage, as this can cause
misalignment. When transporting it, use a microscope bag.
Tip 2: Keep lenses clear of slides
When using your microscope and adjusting the focus you will need to lower the objective lens down
as far as it will go. However, you should never allow the lens to touch the slide you are looking at.
Dirty lenses can be difficult to clean.
Tip 3: Clean after using immersion oil
If using immersion oil, always ensure the objectives are cleaned immediately after use. Objective,
eyepieces and condenser may be removed for cleaning. Use only lens paper and lens cleaner. Do not
use solvents.
Tip 4: Cover when not in use
All microscopes are sold with dust covers. Always keep your microscope covered when not in use
even if the microscope is stored in a cabinet. Eye tubes also need to be kept free of dust so do not store
a microscope without the eyepieces. If the microscope eyepieces must be removed, cover the tubes
with caps or a plastic bag with a rubber band around the eye tube.
Tip 5: Look after the bulb
After using the microscope, turn off the illuminator and wait for it to cool for several minutes before
putting it away. By allowing the bulb to cool you will extend its life. When turning the microscope on
and off, use the switch not the power point. Do not switch the microscope on while using full light
intensity. Never touch the bulb with your fingers as the body oils can burn into the bulb and reduce its
life. Use a tissue. Keep a store of replacement bulbs and always use the correct bulb.

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Tip 6: Store in a clean, dry place
Make sure you do not store your microscope in an area that has corrosive chemical fumes that can
destroy lenses or metal parts or beside solutions that may leak. Salt air and pervasive damp can also
cause damage over time. Make sure your cabinet is ventilated.
Tip 7: Only use special lens paper or wipes for cleaning the lenses
Microscope lenses can easily be scratched and should be treated with great care. Use an aspirator to
remove dust. Sticky residue can be removed with lens paper moistened with distilled water or lens
cleaning solution and rubbed gently using a circular motion. Never use sharp instruments or anything
abrasive on the microscope lenses.
Tip 8: Keep your User's Manual and wrenches in a safe place
Each microscope should come with a user's manual and specialist wrenches as required. Always
consult the User's Manual before making any adjustments to your microscope and use the wrenches
provided. Never over-tighten or use force when performing any maintenance on your microscope, or
use inappropriate tools. This can damage the parts.
Tip 9: Perform an annual maintenance check
On an annual basis moving parts on the microscope should be cleaned and lubricated. Clean grease
and dirt from sliding surfaces using a clean cloth. Apply a very thin layer of lithium-based grease to
the sliding surfaces. Do not grease the teeth of the rack and pinion gears. Inspect the power cords and
plugs for safety and stock up on a supply of replacement bulbs.
Tip 10: Have your microscope serviced professionally
A rule of thumb for frequency of servicing is every 200 hours of use or every 3 years, whichever comes
first.

Application:
• In pathology labs, a compound microscope is extremely useful for detecting illnesses.
• Human cells are drawn and examined under a microscope in forensic laboratories to detect and
solve various crime cases.
• Compound microscopes can be used to detect the presence or absence of minerals as well as
the presence of metals.
• The employment of a microscope in academic activities by students in schools and colleges
has proven to be beneficial.
• It helps in the visualization and comprehension of the microbiological world of bacteria and
viruses, which is otherwise undetectable to the naked eye.

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 Practical 3

Aim: Study of Zooplankton and Phytoplankton from river/pond/lake/estuaries water sample

using compound microscope (Temporary mounting).

Background Information:
Plankton are the mostly microscopic plants and animals that drift in the currents. Plant plankton is
called “phytoplankton,” while animal plankton is called “zooplankton.” Plankton form the basis of life
in the sea. Plankton are the plants and animals that drift around on the oceans’ currents. They are
abundant in the surface waters where sunlight and nutrients are readily available. Phytoplankton are
the microscopic plants that convert sunlight and nutrients to starch and organic matter. Not only do
phytoplankton form the base of the oceans’ food chain, they also produce at least 80% of the oxygen
that we breathe. Among the animal plankton (zooplankton), common are eggs, larvae, and juvenile
forms of invertebrates and fishes. Copepods (related to crabs and shrimp) are the most abundant and
widely distributed zooplankton. All forms of life in the ocean depend either directly or indirectly upon
plankton for food.
Phytoplankton supports most of the herbivores of the sea. As the oceans’ primary producer,
phytoplankton trap and store the energy contained in sunlight. In the process of photosynthesis, the
phytoplankton use carbon dioxide and water to produce food. Because they need sunlight to
photosynthesize, phytoplankton are generally found near the surface of the ocean. Some scientists
calculate that without the oxygen production of phytoplankton, life as we know it would not exist. This
is because phytoplankton produce oxygen in proportion to the amount of carbon dioxide that they use.

Requirements:
Compound microscope, Dissecting microscope, Water sample, Petri dish, Microscope slides, Eye
dropper, Plankton Identification Chart.

Procedure:
1. Use an eye dropper to collect a few drops of the sample and place in a petri dish. Observe the
sample with a dissecting microscope. Since the plankton can move up and down in the drop,
you will need to refocus your microscope to see plankton at different levels.
2. Many of the organisms are too small to be seen with the dissecting microscope. You can prepare
a slide of the sample and observe it under a compound microscope with low and highpower
objectives. Make sure that you save all of the sample.
3. To prepare the students to distinguish different plankton types, you may wish to discuss how
to differentiate ocean plants from animals. Will they have different colors? Structure?
Behavior? You may want to alert them that distinguishing characteristics for land-based plants
and animals are usually much different than those for plankton.
4. Observe your sample for the following: a. most abundant organisms b. variations in shape,
color, and swimming ability c. types of appendages seen on various plankton d. eggs e. larval
and juvenile forms of crustaceans and fish.
5. Select the most common organisms from your sample. Record the following information on
your data sheet: a. a detailed drawing of the specimen b. identification of your specimen using
the Plankton Identification Chart.

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 6. Repeat this procedure with as many different organisms as time permits. Make sure that you
have at least: a. two different kinds of phytoplankton (plants) b. four different kinds of
zooplankton (animals) c. one diatom, one dinoflagellate, and one form of permanent
zooplankton.

Observation:

Sr. No. Name of the species Zooplankton/ Phytoplankton Description

Common name
Scientific name
Family
Habitat
Salient features

Observation Chart:

PHYTOPLANKTONS:

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Page 14 of 38
ZOOPLANKTONS:

Page 15 of 38
Result:

Conclusion:

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 Practical 4

Aim: Separation of lipids in a given sample by TLC.

Background Information:
Thin layer chromatography (TLC) is a tried-and-true method for the separation of components in a
mixture based on the polarity of the individual components. When a standard is included, this method
can also be used for the identification of each component of the mixture.
The TLC technique requires 2 phases: a stationary phase, which involves some sort of matrix, and
a mobile phase, which is a mixture of solvents. The type of mixture you are interested in separating
dictates the precise materials used for the 2 phases.
The stationary phase for TLC consists of a plate, usually a sheet of plastic or glass, that is coated with
an absorbent material (i.e. silica). The extracted sample mixture (such as a lipid extract from cells) is
applied at the bottom of the plate, allowing the solid phase to capture the mixture. The plated sample
is then “developed” by placing the TLC plate into a sealed chamber containing the mobile phase
solvent system. The solvent system is selected based on its ability to dissolve the desired components
to be separated. This chamber also contains a Whatman Paper “wick,” which is a sheet of filter paper
that helps to ensure that the chamber is evenly saturated with solvent vapors.
As the solvent migrates up the plate, it will carry with it the different components of the mixture. The
more soluble the components in the solvent, the easier it will migrate up the plate. Also affecting how
the samples migrate is the absorbent material on the plate. For instance, silica, which is polar, will
strongly interact with the polar components of the mixture. Therefore, polar components will “stick”
to the silica, and will migrate up the plate with less efficiency than components that are weakly polar
or nonpolar. In general, as the polarity of a component (molecule) decreases, the ability for it to move
up the plate increases.
For the purposes of lipid separation, particularly for neutral lipids such as triglycerides, we will be
using a silica coated plastic plate (stationary phase) and an organic, largely nonpolar solvent mobile
phase consisting of petroleum ether: diethyl ether: acetic acid at a ratio of 80:20:1. The lipids are then
visualized using resublimed iodine, which will bind to double bonds found in lipid hydrocarbon chains
and aromatic compounds.
Requirement:
1. Activated TLC plates: Place thoroughly cleaned and dried glass plates (20x20 cm) and spread a
uniform layer of (0.2mm thickness) Silica Gel-G slurry with the help of a spreader, dry at room
temperature and then activate at 110 °C for 30 min.
2. Thin layer chromatographic tanks:
3. Developing Mixture/solvent system: petroleum ether:diethyl ether:glacial acetic acid (80:20:1 v/v)
4. Spraying reagents for location of spots on TLC plates: 50% sulphuric acid.
5. Lipid standards: cholesterol, palmitate, lecithin etc.,

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Principle:
This technique is similar to paper chromatography but is more convenient and less time consuming.
Here instead of paper, the supporting material is either a glass plate or a plastic sheet or a piece of
metal foil. A thin layer of stationary i.e., silica gel (SiO2) or alumina (Al2O3) is laid over this inert
support. The solvent system is selected according to type of biomolecule under investigation.

Procedure:
1. Take an activated TLC plate and draw two straight lines. First one about 2 cm from the bottom
and second one 1 cm from the top of the plate.
2. Subdivide the bottom line for spotting the samples with 2 cm between two samples.
3. Pipette 20 µl of all the standard and test samples, and spot in an order on the TLC plates. Air
dry the plates for 5-10 min.
4. Meanwhile add solvent (mobile phase) to the TLC chamber and close it with the lid. Allow it
to saturate the chamber for 10 min at room temperature.
5. Using forceps, pickup the TLC plate from the top; place the TLC plate in the TLC chamber
vertically. Ensure that the solvent phase moves uniformly along the plate.
6. Leave the plate in the chamber until the solvent has moved to the top pencil line of the TLC
plate. When the solvent front has moved to the top line remove the plate with the help of
forceps.
7. Place the TLC plate on a clean dry surface or on tissue and allow the mobile phase to evaporate
completely for about 5-10 min.
8. Spray the detection reagent carefully and heat it in an oven at 110 °C for 5-10 min. areas
containing lipids get charred and appear as black spots.
9. Locate the position of the lipid spots on the plate and measure the distance travelled by the
individual lipid component.

Calculate the Rf value of each sample, identify and report the lipids present in the sample given.
Rf = Distance travelled by solute
Distance travelled by solvent

Calculation:

Observation Table:

Sr. No Sample Rf Value

1. Standard (Cholesterol)
2. Standard (Lecithin)
3. Sample 1

Result:
The given standard No. 1 has the Rf value of ---- (a) and standard No. 2 has the Rf value of ----(a1).
The given sample No. 1 has the same Rf value of standard No. 1. Hence the sample No. 1 might be
cholesterol.

Conclusion:

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 Practical 5

Aim: Separation of pigments from leaves or flowers by adsorption column chromatography.

Background Information:
Column chromatography is a technique which can be applied to the separation of many complex
mixtures. The sample solution is applied to the top of the column. The mobile phase flows down
through the column filled with the stationary phase material.

Requirement:
Chromatography column, glass wool cotton wool, beaker, conical flask, mortar china dish, calcium
carbonate, anhydrous sodium sulphate, anhydrous, benzene, petroleum ether, ethyl alcohol, separatory
funnel, graduated cylinder and wash bottle.

Principle:
The success of a separation by column chromatography depends on the choice of the stationary and
mobile phases. The stationary phase material is filled in a column. Any of the three possible
mechanisms: partition, adsorption or ion exchange can be employed by the use of a particular type of
the stationary phase inside the column. For example, for the separation based on adsorption an
adsorbent is packed in the column. The choice of the mobile phase depends on the nature of the
substance and how strongly it is adsorbed. In a number of cases such as alumina and silica gel as the
adsorbent, the mobile phase is generally a non-polar solvent such as petrol and benzene because polar
groups such as hydroxyl-(OH) group in water and in ethanol would cause desorption. Eluents
containing two or more solvents may be used for better results. In such cases the polarity is increased
by adding a polar solvent with a Column Chromatographic Separation of Pigments From non-polar
one.

Procedure:
1. Preparation of the Extract:
1) Take 5-10 g of fresh grass (or leaves of a green plant) cut it up into fine pieces in a mortar,
grind for about 30 seconds, add 1 10 cm of ethyl alcohol and 20 cm3 of petroleum ether, grind
again.
2) Decant the liquid into a separatory funnel after filtering through glass wool placed in an
ordinary funnel.
3) Add 10 cm3 alcohol and 20 cm3 petroleum ether again to the mortar containing grass, grind
and transfer the liquid after decantation to the separatory funnel containing the first fraction.
4) Shake gently. A light green emulsion may form, if shaken vigorously.
5) Allow to settle the layers.
6) The solution and solvent bottom layer is water-ethanol layer and the upper layer of petroleum-
ether contains grass extract. Remove the bottom layer and wash the petroleum layer with water
for 3 or 4 times until the layer is clear.
7) Remove the aqueous layer. Glass wool the extract is now free from alcohol but contains water
in very small amount. Transfer the upper layer containing the extract to a dry conical flask.

Page 19 of 38
 8) To this, add anhydrous sodium sulphate (dried by heating in an oven hot plate before use),
shake the flask and leave it over for about 15 minutes to remove any water present with the
extract.
9) Transfer the extract to a clean and dry test tube, cover it and take it for chromatography.

2. Preparation of column:
1. Take a glass column or a burette of about 20 cm in length and 7-8 mm diameter tube.
2. Place a small wad of cotton wool as the column support.
3. Pack the column with anhydrous calcium carbonate (dried by heating in a china dish over a
burner), tap it regularly with a glass rod.
4. Add the adsorbent in small portions and gently press down until a column of 8-10 cm has been
uniformly packed.
5. Place a small wad of cotton wool at the top of the calcium carbonate column and use it for
chromatography.
3. Take the uniformly packed column containing calcium carbonate and fix it in a stand vertically.
4. Take 1-2 cm3 of dried extract of leaves, drip into the column in the form of a thin layer of solution,
allow to run evenly into the adsorbent unit: a green zone 3-4 mm deep is formed at the top of the
column. This is known as the loading of the sample.
Chromatographic column: The physical state of the column packing material should be such that it
allows uniform packing of the column and a free flow of the solvent through it. The extract from green
leaves should be completely free from water since the presence of a polar substance can alter the course
of separation.
5. Add the developer (benzene) to the column and allay the developer through the column packing till
separate bands are observed.
6. Note the colour of different bands and their order.
7. If extra time is available, continue the passage of developer and collect the different coloured
substances in fractions, noting the volume eluted by a measuring cylinder.

Result:
The bands observed on the column are of different colours. The uppermost thin yellowish green zone
is chlorophyll-b, below this the bluish green zone of chlorphyll-a, next orange-yellow zone contains
xanthopylls and the lowest orange zone contains carotenes. The carotenes are least adsorbed by the
adsorbent and can be easily washed out of the column. Three main interactions are to be considered in
column chromatography: the activity of the adsorbent, the polar behaviour of the substance and the
polarity of the eluting solvent. This experiment is based on the results of the inventor of the technique
of chromatography, M. Tswett, who applied the technique to separate various plant pigments using
calcium carbonate as the stationary phase packed in a column.

Conclusion:

Page 20 of 38
 Practical 6

Aim: Separation and identification of amino acids by 2D paper chromatography.

Background information:
Chromatography is used to separate mixtures of substances into their individual components. All forms
of chromatography work on the same principle. They all have basic requirements of stationary phase
(a solid or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows
through the stationary phase and carries the components of the mixture with it. Different components
travel at different rates based on their affinities toward stationary phase and mobile phase. In paper
chromatography, the stationary phase is a very uniform adsorbent paper. The mobile phase is a suitable
liquid solvent or mixture of solvents. Retention (or) retardation factor (Rf)- Retention factor is defined
the ratio of the distance travelled by the solute to the distance travelled by solvent. The distance
travelled relative to the solvent is called the Rf value.

Rf = Distance travelled by solute

Distance travelled by solvent

Requirement:
Apparatus: Glass beakers Whatmann filter paper, Petri dishes, Measuring cylinder, Developing
chamber and capillary tubes etc.
Chemicals: n-butanol Glacial acetic acid Distilled water (4:1:5) Amino acids (Tryptophan and
threonine) Ninhydrin reagents. Solvents system and its preparation methods 1. n-butanol and water are
taken in 4:5 ratios in a conical flask and allow it to saturate for 24 hours. By using separating funnel
separate nbutanol and water. The saturated n-butanol and Glacial acetic acid are ta ken in the ratio of
4:1 which can be used as a solvent system (or) mobile phase.

Principle:
In paper chromatography separation of component is distributed between phases of liquid. Here, one
phase of liquid is water that is held amidst the pores of filter paper and the other liquid is the mobile
phase that travels along with the filter paper. Separation of the mixture is the result that is obtained
from the differences in the affinities towards the water and mobile phase when travelling under
capillary action between the pores of the filter paper.

Procedure:
Paper chromatography
1. The procedure for paper chromatography method is quite simple as compared to other methods
of chromatography.
2. The chromatography paper is cut into rectangular strips and marks a line on the paper with
pencil at about 2 cm from the bottom.
3. With the help of capillary tube, the samples are applied at different points on the starting line.
Now, place the chromatography paper in the developing chamber, which contains the mobile
phase.
4. While placing the paper, it is important that the solvent level should not reach the starting line
or the sample spots and paper shouldn’t touch the walls of the developing chamber.

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 5. After sometime the solvent rises up the paper or the stationary phase by capillary action and
dissolves the sample. The components of the sample move along with the solvent in upward
6. direction. Check if the solvent has reached near the top level of chromatography paper.
7. Then the paper is removed when it reaches the top and marked the level with pencil. This level
(or) height is called the “solvent front”.
8. By using UV light, ninhydrin or iodine vapors examined the different spots of varied colors.
Each spot represents a specific component of the sample.
Result:
Rf value of the given sample is__________.

Conclusion:
For paper chromatography the distance moved by tryptophan and threonine is ______cm and ____ cm
respectively, and the solvent is _____cm.
Rf value of tryptophan is _____Rf value of threonine is ____Rf value of unknown mixture is____ &
____By performing the paper chromatography, the distance moved by the sample and unknown
mixture is noted and by substituting these values in the given Rf formula, the Rf values of tryptophan,
threonine and unknown samples are known.

Page 22 of 38
 Practical 7

Aim: To study the following instruments through photographs.

1. Spectrophotometer

Ultraviolet/Visible area (UV-Vis) measurements span wavelengths from around 200 nm to 800 nm.
The absorption by a molecule of ultraviolet or visible radiation results in transitions between the
molecule’s electrical energy levels. The optical and electronic properties of different materials, such
as films, powders, monolithic solids, and liquids, are suitable for characterization.

UV-vis spectroscopy is a cost-effective, simple, versatile, non-destructive, analytical technique

suitable for a large spectrum of organic compounds and some inorganic species. As a function of
wavelength, UV-vis spectrophotometers measure the absorption or transmission of light that passes
through a medium.

Ultraviolet-visible (UV-Vis) spectrophotometers use a light source to illuminate a sample with light
across the UV to the visible wavelength range (typically 200 to 800 nm). The instruments then measure
the light absorbed, transmitted, or reflected by the sample at each wavelength. Some
spectrophotometers have an extended wavelength range, into the near-infrared (NIR) (800 to 3200
nm).

Application of UV-Vis spectrophotometer:

• Identify molecules in a solid or liquid sample.
• Determine the concentration of a particular molecule in solution.
• Characterize the absorbance or transmittance through a liquid or solid—over a range of
wavelengths.
• Characterize the reflectance properties of a surface or measure the color of a material.
• Study chemical reactions or biological processes.

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 2. Flame photometer:

During 1980s Bowling Barnes, David Richardson, John Berry and Robert Hood developed an
instrument to measure the low concentrations of sodium and potassium in a solution. They named this
instrument as Flame photometer. The principle of flame photometer is based on the measurement of
the emitted light intensity when a metal is introduced into the flame. The wavelength of the colour
gives information about the element and the colour of the flame gives information about the amount
of the element present in the sample. Flame photometry is one of the branches of atomic absorption
spectroscopy. It is also known as flame emission spectroscopy. Currently, it has become a necessary
tool in the field of analytical chemistry Introduction: Flame photometer can be used to determine the
concentration of certain metal ions like sodium, potassium, lithium, calcium and cesium etc. In flame
photometer spectra the metal ions are used in the form of atoms.
Applications of Flame Photometer:
• Clinical Chemistry: Measurement of electrolytes, such as sodium and potassium, in blood or
urine samples.
• Environmental Analysis: Determination of alkali metals in soil, water, and plant samples.
• Pharmaceutical Analysis: Quantification of trace elements in pharmaceutical formulations.
• Food and Beverage Industry: Analysis of mineral content in food and beverages for quality
control purposes.
• Geological Studies: Measurement of elements in geological samples for mineral exploration
and research.

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 3. SEM

Scanning Electron Microscope (SEM) is a type of electron microscope that scans surfaces of
microorganisms that uses a beam of electrons moving at low energy to focus and scan specimens. The
development of electron microscopes was due to the inefficiency of the wavelength of light
microscopes. electron microscopes have very short wavelengths in comparison to the light microscope
which enables better resolution power.
The first Scanning Electron Microscope was initially made by Mafred von Ardenne in 1937 with an
aim to surpass the transmission electron Microscope. He used high-resolution power to scan a small
raster using a beam of electrons that were focused on the raster. He also aimed at reducing the problems
of chromatic aberrations images produced by the Transmission electron Microscopes. More studies
followed by scientists and research institutions such as Cambridge Scientific Instrument Company
who eventually developed a fully constructed Scanning electron Microscope, in 1965 and named it a
Stereoscan.

Application of Scanning Electron Microscope

1. Used for spot chemical analysis in energy-Dispersive X-ray Spectroscopy.
2. Used in the analysis of cosmetic components which are very tiny in size.
3. Used to study the filament structures of microorganisms.
4. Used to study the topography of elements used in industries.

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 4. TEM

Transmission Electron Microscope (TEM): This is a powerful electron microscope that uses a beam
of electrons to focus on a specimen producing a highly magnified and detailed image of the specimen.
The magnification power is over 2 million times better than that of the light microscope, producing
the image of the specimen which enables easy characterization of the image in its morphological
features, compositions and crystallization information is also detailed. Early discovery of cathode rays
like electrons by Louis de Broglie in the early 1920s, paved way into the development of an electron
microscope where they used a beam of electrons creating a form of wave motion. Magnetic fields were
used as lenses for the electrons. With these discoveries, the first electron microscope was later
developed by Ernst Ruska and Max Knolls in 1931 and modified into a Transmission Electron
Microscope (TEM) by Ernst Ruska along with the Sieman’s company, in 1933.This TEM microscope
has several advantages compared to the light microscope with its efficiency also being very high.
Among all microscopes both light and electron microscopes, TEM are the most powerful microscopes
used in laboratories. It can magnify a mall particle of about 2nm, and therefore they have a resolution
limit of 0.2um.

Application of Transmission Electron Microscope

1. To visualize and study cell structures of bacteria, viruses, and fungi
2. To view bacteria flagella and plasmids
3. To view the shapes and sizes of microbial cell organelles
4. To study and differentiate between plant and animal cells.
5. Its also used in nanotechnology to study nanoparticles such as ZnO nanoparticles
6. It is used to detect and identify fractures, damaged microparticles which further enable
repair mechanisms of the particles

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 5. HPLC

High Performance Liquid Chromatography (HPLC) is a method in analytical chemistry used to

isolate the components in a mixture and to identify and quantify each component. It was first
discovered as an analytical technique in the initial twentieth century and was used to isolate colored
compounds in a mixture. The word "chromatography" refers to "colour writing." He was the botanist
M. S. Tswett who invented this technique in around 1900 to study leaf pigments (mainly chlorophyll).
He isolated the pigments based on their interaction with a stationary phase. In 1906, Tswett invited
two fundamental documents describing the various characteristics of liquid
adsorption chromatography in detail. He also pointed out that, in spite of its name, other substances
can also be separated by chromatography. Recently high-performance liquid chromatography has
developed from the separation efficiency, versatility, and speed has been improved significantly. The
molecular species subjected to separation exist in a sample that is composed of analytes and matrix.
The analytes are the molecular species of concentration, and the matrix is the rest of the components
in the sample. HPLC can only evaluate chemicals that are dissolved in solvents. HPLC separates
chemicals dispersed in a liquid sample, allowing for qualitative and quantitative examination of which
components are present in the sample and how much of each component is present. In the 1960s, LC
with low-pressure glass columns evolved into high-pressure chromatography (HPLC) using metal
columns. As a result, it is a better version of TLC. Instead of allowing a solvent to drop through a
column under gravity, it is pushed through at up to 400 atmospheres of pressure.

Application of HPLC:
• Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics
• Determination of drugs in biological matrices
• 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
• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides

Page 27 of 38
 6. GCMS

Gas chromatography mass spectrometry (GC-MS) is an analytical technique that combines two
powerful techniques; gas chromatography and mass spectrometry and is used to separate, identify, and
quantify volatile compounds. It is therefore perfect for analyzing the many relatively low molecular
weight compounds. Although it can be used with solid, gaseous, and liquid samples, GC-MS analysis
is most often used with volatile and semi-volatile compounds. In Gas Chromatography (GC), the
chemical constituents of a sample mixture are separated, which is then used to identify the components
to ascertain whether or not they are present, as well as how much of each component is present. The
information provided by GC detectors is constrained; it is typically two-dimensional and includes the
retention time on an analytical column and the detector response. Identification is based on comparing
the retention times of the peaks in a sample to standards of well-known substances that were also
subjected to the same kind of analysis. Although GC alone cannot be utilized to identify unknowns,
hyphenation to mass spectrometry can be quite effective in such a scenario. The molecular weight and
elemental constituents of molecules, as well as their chemical structures, can be determined using the
analytical method known as MS, which measures the mass-to-charge ratio (m/z) of charged particles.
A GC-MS produces three-dimensional data, including chromatograms for qualitative and quantitative
examination as well as mass spectra for confirming the identity or identifying chemical compounds.

Application of GC-MS:
Medicine: Several congenital metabolic illnesses can be detected with the help of GC-MS screening
techniques. Patients with hereditary metabolic abnormalities can be diagnosed by testing their urine
for the presence of trace chemicals.
Biological and pesticide detection: Blood and urine can be tested for the presence of drugs using GC-
MS. This includes anesthetics, anticonvulsants, antihistamines, sedative-hypnotics, opioids, and anti-
epileptic medications.
Pharmaceutical industries: The pharmaceutical industry makes extensive use of GC-MS in the areas
of analytical development, quality control, quality assurance, production, and pilot plants for API, bulk
medicines, and formulations.
Clinical toxicology: GC-MS is extensively employed in clinical toxicology to identify toxins and
venoms.
Environmental monitoring: The gas chromatograph-mass spectrometer (GC-MS) is now widely used
as a method for detecting organic contaminants in the environment.

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 Practical 8

Aim: Colorimetric estimation of serum/egg protein by Peterson‐Lowry method.

Background information:
Lowry assay is one of the old methods used for protein estimation developed by Oliver Lowry (1951).
This is an overly sensitive and gives accurate results. It detects proteins at low concentrations of 2-5
µg. In this method, first, the copper ions are reduced under alkaline conditions and forms a complex
with peptide bonds of the protein. This complex then reduces Folin-Ciocalteau reagent and results in
the change in color to deep blue and absorption can be measured at 650-750nm. It takes about 45-60
minutes for assay completion. A Compatible Lowry Assay for samples with reducing agents and other
interfering agents and a Modified Lowry Assay for samples in detergents are available.

Requirement:
1. Reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide.
2. Reagent B: 0.5% copper sulphate (CuSO4.5H2O) in 1% potassium sodium tartarate. Prepare fresh
by mixing stock solutions.
3. Alkaline copper solution (Reagent C): Mix 50mL of reagent A and 1 mL of reagent B prior to use.
4. Diluted Folin’s reagent (Reagent D): Dilute Folin-Ciocalteau reagent with an equal volume of 0.1
N NaOH
5. Standard: Dissolve 50mg BSA in 50mL of distilled water in a volumetric flask. Take 10mL of this
stock standard and dilute to 50 mL in another flask for working standard solution. One mL of this
solution contains 200 µg protein.
6. Apparatus and Glass wares required: Test tubes, Pipettes, Colorimeter, etc.
7. Sample: Serum/Egg protein

Principle:
The –CO-NH- bond (peptide) in polypeptide chain reacts with copper sulphate in an alkaline medium
to give a blue colored complex. In addition, tyrosine and tryptophan residues of protein cause reduction
of the phosphomolybdate and phosphotungstate components of the Folin-Ciocalteau reagent to give
bluish products which contribute towards enhancing the sensitivity of this method.

Procedure:
1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of labeled test tubes.
2. Pipette out 1 mL of the sample in another test tube.
3. Make up the volume to 1 mL in all the test tubes. A tube with 1 mL of distilled water serves as the
blank.
4. Now add 5 mL of reagent C to all the test tubes including the test tubes labeled 'blank' and 'unknown'.
5. Mix the contents of the tubes by vortexing / shaking the tubes and allow to stand for 10 min.
6. Then add 0.5 mL of reagent D rapidly with immediate mixing well and incubate at room temperature
in the dark for 30 min.
7. Now record the absorbance at 660 nm against blank.
8. Then plot the standard curve by taking concentration of protein along X-axis and absorbance at 660
nm along Y-axis.
9. Then from this standard curve calculate the concentration of protein in the given sample.

Page 29 of 38
Result:
The given unknown sample contains_______µg protein/ml.

Conclusion:

Page 30 of 38
 Practical 9
Agarose Gel Electrophoresis (AGE)

Aim: To analyze the given nucleic acid using Agarose Gel Electrophoresis.

Background Information:
Agarose gel electrophoresis is routinely used in molecular biology and genetic engineering for the
visualization, purification and characterization of DNA molecules. DNA molecules are negatively
charged and therefore migrate through the agarose gel matrix to the positive terminal at the bottom of
the gel. Electrophoresis is used in many aspects of science for the separation of charged molecules,
whether they are RNA, DNA or proteins. For the separation of nucleic acids, a compound purified
from seaweed, agarose, is routinely used. Agarose, a linear polymer, is dissolved in hot running buffer
and on cooling it polymerizes into a semi‐ solid matrix. The polymerization process of agarose
involves the sugar groups cross‐ linking to form a matrix. The resulting matrix allows small molecules
to move, or migrate, quickly through the gel, whereas the larger molecules are hindered by the matrix,
causing them to migrate slower. This principle allows the separation of molecules by size. The
addition of an electric current to the gel causes the molecules to move in one direction, towards the
positive end of the gel.

Requirement:
1. Agarose solution,
2. Ethidium bromide,
3. Electrophoresis buffer, 4. 6x gel buffer,
5. DNA sample,
6. DNA size standard.

Principle:
Agarose gel electrophoresis used to analyze and quantitate nucleic acid. The Agarose for Agarose gel
electrophoresis is purified from agar. Agarose is a linear polymer made up of repeating units of 1,3 –
linked ß D galactopyranose and 1, 4 linked 3,6 anhydro a L galactopyranose [ P-D –gal (1-4)-3,6
anhydro – a L Gal (1-3) -] n Agarose has an average MW of 12,000 and contains about 35-40
agarobiose units. Agarose in solution exist as left-handed double helices. About 7 to 11 such helices
form bundles which extend as long rods and appear to intertwine with one another, further
strengthening the frame work of the gel. The cross links are held together by hydrogen and
hydrophobic bonds. By changing the gel concentration, the pre size can be altered. Higher the
concentration of Agarose smaller the pre size and vice versa. Because of large pore size even at low
concentration, Agarose gels are widely used for separation of DNA and RNA.

Preparation of stock solutions for DNA gel electrophoresis:

To different buffer systems are widely used for separation of nucleic acids by agarose gel
electrophoresis. Their composition are given in the table:

Page 31 of 38
Sterilize the stock solutions by autoclaving

Preparations of ethidium bromide (stock solution)

Weigh 10 mg ethidium bromide into a sterile tube and dissolve in 10 ml sterile distilled water. The
stock is stored at 4°C
Preparation of sample loading dye Glycerol & bromophenol blue (6x)
3ml glycerol (30%), 25mg bromophenol blue (0.25%) dH2O to 10mL
Preparation of agarose solution for casting the gel
Dissolve the Agarose by placing the flasks in boiling water both cool to Luke warm. Cover the sides
of a tray using cellotape and place the comb about 1 cm from the top of the tray. Pour the Agarose
without making any bubbles, cool it for 20 mins and take off the combs and uncover the cellotapes

1. The DNA sample (100 to 200 ng) is mixed with the loading dye (for 5 µl of DNA sample 1µof
6x dye is used) and loaded in to the well carefully, using a pipetman or capillary tube.
2. Once the sample is loaded in to the well, the cathode (Black negative terminal) is connected
towards the top end of the gel and the anode (Red positive terminal is connected towards the
bottom end of the gel.
3. The maximum volume that can be loaded on to a well formed from a 1.5 mm thickness tooth
of the comb is 30 µl. The electrophoresis is started by switching on the D. C. Powerpack. The
gel is run at 5v/cm.
4. As the bromophenol blue(the tracking dye) has moved 1 cm above the bottom end, the current
is switched off, the power supply is disconnected and the gel along with the platform is stained
in the plastic tray containing 0.5 µg/ml ethidium bromide in the sterile distilled water( use
gloves when handling ethidium bromide).
5. After about 30-45 min, the platform and the gel is rinsed with distilled water and by keeping
the platform in a slanting position, the gel is gently pushed onto the UV Transilluminator. (As
UV rays are dangerous for the eye, protect your eyes by wearing a UV face shield, goggles or
using glass plate).

Page 32 of 38
 6. Now the UV light is switched on and the DNA bands are seen and Photographed at f 5.6 for 10
seconds with an orange filter.

Phenol: Chloroform Extraction:

1. Mix the DNA solution with equal volume of phenol: chloroform (1:1 v/v).
2. Centrifuge at 10,000 rpm 5 min.
3. Transfer the aqueous phase to a fresh tube, add equal volume of chloroform, and centrifuge 10,000
rpm for 5 min.
4. Transfer the aqueous phase to a fresh tube and mix with 1/10th volume of 3 M NaOAc and 2.5
volume of ethanol. Leave at -20°C for 1 h for precipitation.
5. Centrifuge the samples at 10,000 rpm for 10 min; decant the supernatant.
6. Add 1 ml of 70% ethanol to the pellet, vortex and centrifuge at 10,000 rpm for 5 min.
7. Air-dry the pellet and dissolved in appropriate volume of 0.1X TE buffer.

Result:
After electrophoresis DNA bands can be visualized under UV light and they appeared as orange
fluorescence.

Conclusion:

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 Polyacrylamide Gel Electrophoresis (PAGE)

Aim: Separation of proteins using SDS-PAGE

Background:
Polyacrylamide gel electrophoresis (PAGE) is a technique use almost universally in life science
laboratories. The goal of this technique is to separate a mixed sample of proteins to identify and
quantify single proteins from the mixture. The starting sample could come from any number of sources
such as a patient sample, homogenised tissue or bacterial culture. It is also possible to use PAGE to
separate DNA and RNA, but proteins are the most common sample type. There are several types of
PAGE technique that are used, but the most common is called SDS-PAGE. In SDS-PAGE the detergent
Sodium dodecyl sulfate is used to denature the proteins and normalise their mass-to-charge ratio.
Without SDS, both the molecular weight and the charge of the protein would affect its separation in
the gel. With SDS, only the molecular weight affects the migration speed and so samples separate
according to this. PAGE without SDS is called native PAGE, as the proteins stay in their native
conformation.
Requirement:
Reagents Required:
1. Preparation of stock solution and buffers: 30% acrylamide
a) Acrylamide: 29.2g
b) N, N-methelyne–bis–acrylamide: 0.8g Added water, dissolved and made upto 100mL and filtered
with Whatman no.1 filter paper.
2. Separating gel buffer:
a) Tris-HCl: 1.5M, pH 8.8 18.171g of Tris was dissolved in 60mL of water and adjusted the pH to 8.8
with HCl and finally made upto 100mL with water.
3. Stacking gel buffer:
a) Tris-HCl: 1M, pH 6.8 6.057g of Tris was dissolved in 60mL water and adjusted the pH to 6.8 with
HCl and upto 100mL with water.
4. 10% SDS solution: 1g of SDS in 10mL of distilled water.
5. N,N,N’N’-Tetra methylene diamine (TEMED)
6. 10% Ammonium per sulphate (APS): 1g of APS in 10mL of distilled water.
7. Electrophoresis Buffer:
a) Tris: 25mM, pH 8.3
b) glycine: 250mM,pH 8.3
c) SDS: 0.1%: Dissolved in minimum amount of water (500mL) and then added SDS. Allowed to
settle and dissolved. This was finally made upto 2.5liters.
8) Sample buffer 4x: 5.0mL
a) Tris (1M, pH 6.8): 2.1mL
b) 2% SDS: 100mg
c) Glycerol (100%): 1.0mL
d) b-mercaptoethanol: 0.5mL
e) Bromophenol blue: 2.5mg
f) Distilled water: 0.4mL
9) Staining solution (100mL):

Page 34 of 38
a) Alcohol: 40%
b) Acetic acid: 10%
c) Commassie Brilliant Blue (CBB): 259mg
d) Distilled water: 50%
10) Destaining solution (100mL)
a) Alcohol: 50%
b) Acetic acid: 10%
c) Distilled water: 40%

Principle:
Polyacrylamide Gel Electrophoresis (PAGE) operates on the principle that charged molecules migrate
in an electric field towards an electrode with the opposite charge. In the case of SDS-PAGE, proteins
are treated with sodium dodecyl sulfate (SDS), an anionic detergent that binds to the proteins and
imparts a negative charge to them. The negatively charged proteins are then loaded onto a
polyacrylamide gel and subjected to an electric field, causing them to migrate towards the positively
charged electrode (anode). The proteins are separated based on their size as they move through the gel
matrix, which acts as a molecular sieve. After the electrophoresis process, the separated proteins can
be visualized using protein-specific staining techniques. By comparing the migration distance of
unknown proteins to that of a known molecular weight marker, the size of the proteins can be
determined. The concentration of acrylamide and the degree of cross-linking in the gel can be adjusted
to control the resolution and separation of different-sized molecules.

Procedure:
Preparation of gel:
1. The glass plates were washed in warm detergent solution, rinsed subsequently in tap water,
deionised water and ethanol and dried.
2. The unnotched outer plates were laid on the table and Vaseline (or grease) was coated. Spacer
strips were arranged approximately at the sides and bottom of the plates.
3. The notched inner plates were laid in position, resting on the spacer strips and the arrangement
was mounted vertically.
4. Sealing was done properly to avoid leakage.
5. The volume of the gel solution required for making separating gel was calculated as follows
(the reagents in the following table yield 20mL of solution after the addition of APS and
TEMED)

Page 35 of 38
6. APS and TEMED were added just prior to the pouring of gel. The solution was mixed well and
poured into the space between the two plates leaving an inch of the upper space unfilled. Water
was carefully laid over the surface of the poured gel mixture to avoid air contact, which reduces
the polymerization reaction.
7. The gel mixture was allowed to polymerize, undisturbed at room temperature for 60 minutes.
In the meantime, gel mixture for stacking gel was prepared. (The reagents in the following
table yield 10mL of solution after the addition of APS & TEMED)
8. After the separating gel was polymerized, the over laid water was removed carefully with filter
paper and an appropriate comb was inserted between the plates. 0.1mL of 10% APS and 10 l
of TEMED were added to the stacking gel mixture. It was mixed well and poured immediately
(to the brim) over the separating gel.
9. The stacking gel was allowed to polymerize. Additional gel mixture was added when gel
retracted significantly.
10. Preparation of protein samples: The required volume of sample buffer was added to protein
samples and they were loaded (the final concentration of sample buffer in the prepared sample
should come to 1x. If the protein was dried suspend it in 1x buffer). The samples were incubated

for 2 min in a boiling water bath prior to loading.

11. When the polymerization was completed, the comb was removed and the lower spacer strip
was carefully removed.
12. The Vaseline (or grease) from the bottom was removed with a piece of tissue paper. The gel
was attached to the electrophoresis tank using appropriate clips/clamps. The lower reservoir
was filled with 1x electrophoresis buffer, using a bent Pasteur pipette or syringe needle to
remove any air bubble trapped beneath the bottom of the gel.
13. The protein samples were loaded using a micropipette and the wells were completely and
carefully filled with 1x electrophoresis buffer. The upper reservoir was also carefully filled
with 1x electrophoresis buffer. The electrodes were connected to a power pack.
14. The gel was run at constant current (20 milli ampere 100 volts) for 4-6 hrs at room temperature.
Electrophoretic mobility of the samples was determined by bromophenol blue front. At the end
of the run the power pack was switched off.
15. The gel and plates were laid flat on the table and a corner of the upper glass plate was lifted up
and the gel was carefully removed. Staining of the gel: After the completion of the
electrophoresis, the gel was fixed with 10% trichloroacetic acid for 5minutes and stained with
CBB.

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 16. The CBB staining solution was prepared using methanol, acetic acid and double distilled water
in the ratio of 4:1:5 and 0.25gm of CBB was added and the gel was stained over night.
17. Destaining of the gel: The destaining of CBB stained gel was done by using methanol, acetic
acid and double distilled water in the ratio of 5:1:4 till the appearance of clear bands on the gel.

Result:
The sample proteins are separated by Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis.
The proteins appeared as discrete bands in the gel. The relative molecular weights of the protein with
respect to their bands were observed in Kilo Daltons.

Conclusion:

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 Practical 10

Field visit to any instrumentation laboratory/research institute/centralized laboratory

Students are expected to submit a certified field visit report to the department/exam center before
commencement of the university practical exam.

Field report should contain following contents:

• General details of field visit: Title, date, day, time, geotagged photo.
• Objectives of field visit
• Introduction of the visiting place
• Observation
• Importance
• Summary
• Learning outcomes

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