I SEMESTER
BIOTECHNOLOGY
MANUAL
G511.1P- BIOPHYSICS
AND BIOSTATISTICS
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INDEX
1. CIRCULAR PAPER CHROMATOGRAPHY
2. ASCENDING PAPER CHROMATOGRAPHY OF AMINO ACIDS
3. SEPARATION OF PHOTOSYNTHETIC PIGMENTS USING THIN LAYER
CHROMATOGRAPHY.
4. DETERMINATION OF ABSORPTION MAXIMUM OF A SOLUTION
5. CYCLOSIS IN HYDRILLA
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1. CIRCULAR PAPER CHROMATOGRAPHY
Aim: To separate and identify carbohydrates in a given mixture by circular paper chromatography
Principle: the cellulose fibres of chromatographic paper acts as supporting matrix for stationary phase.
(Water- polar).Separation is achieved as a result of the related solubility to the (the affinity of substance
to the) stationary and mobile phase. The compounds in am mixture will have the different partition
coefficient. The more polar compounds move less fast as they have greater affinity to the stationary
phase. The less polar compounds move faster as they have greater affinity towards the non-polar
solvent. The fundamental measurement of chromatography is the Rf value (retardation factor) which is
defined as
Rf = distance traveled by the solute
Distance traveled by the solvent.
Requirements:
Whatmann filter paper, petridishes, spraying reagent, solvent(butanol:acetic acid: water)
Preparation of sample: The samples are prepared by dissolving 1 g of each in 100ml of distilled water.
Preparation of the solvent:
Butanol : acetic acid : water
60 : 15 : 25
The solvent is taken in a larger petridish. The petridish is saturated with solvent vapour keeping them closed
overnight with solvent.
Spraying reagent: aniline diphenyl amine
Procedure:
1. The solvent is poured in petridishes and kept closed for saturation overnight.
2. The filter paper is cut to a round shape marking the circumference using the petriplates.
3. The mid point of the filter paper is marked.
4. A small circle is drawn at the centre.
5. The standard samples and the mixture of sample is applied on different spots (4 points) at equidistance in
the inner circle.
6. The sample applied is dried using a hair drier.
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7. The filter paper wick is used to connect the paper with the solvent.
8. The apparatus is kept closed for 1- 1½ hour for the development of chromatogram.
9. After 1½ hour the distance traveled by the solvent (solvent front) is marked with a pencil in the
chromatogram.
10. The chromatogram is dried in a hot air oven or using a hair drier.
11. After drying the chromatographic paper is sprayed with the spraying reagent(aniline diphenyl amine)
12. The paper is dried in the oven and the development of colour is observed.
13. The distance traveled by the solvent and the solute is noted.
14. The Rf value is calculated using the formula:
Rf = Distance traveled by the solute
Distance traveled by the solvent.
Result: The given mixture contains ______________ and ______________ with an Rf value of
____________ and _____________.
Observation and calculation:
Rf = Distance traveled by the solute
Distance traveled by the solvent.
samples distance traveled by distance traveled by Rf value
the solvent the solute
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2. ASCENDING PAPER CHROMATOGRAPHY OF AMINO ACIDS
Aim: to separate and identify amino acids in a given mixture by Ascending paper chromatography
Principle: Cellulose fibres of the chromatography paper acts as the supporting matrix for the stationary
phase, which may be water. The stationary phase is more polar and remains bound to the filter paper
while the mobile phase advances. The stationary phase consists of water in this case and mobile phase
consists of butanol. Amino acids are separated depending on their affinity to either of the two phases (on
the basis of their partition or distribution co-efficient)
Reagents:
Solvent system: Butanol: acetic acid: water
Standard solutions of amino acids
Visualizing agents- 1% Ninhydrin solution in acetone.
Procedure:
1. Take a Whatmann filter no. 1 filter paper which is rectangular shaped
2. Draw a line at a short distance of about 2cm from one edge of the filter paper.
3. Mark 4 equidistance spots
4. Note against each spot the name of the amino acid to be applied on the spot.
5. Apply with the help of capillary tube.
6. The solvent is taken in a chromatography chamber and kept closed for saturation one and a half hour.
7. After the material has been spotted on the filter paper, the paper is folded into a cylindrical form and
pinned at two ends.
8. The folded cylindrical form of filter paper is then immersed into the solvent so that the paper just
touches the solvent. The chamber is then kept closed for one hour for the solvent front to reach the
other end.
9. After one hour, the paper is taken out , the solvent front is marked with a pencil and the paper is
allowed to dry.
10. Visualizing agent i.e. Ninhydrin is sprayed on the paper using a sprayer and then the paper is dried in
a hot air oven at 65º C for few minutes till the colour develops.
11. The position of spots given by individual amino acids is compared with the spots developed from the
mixture. The relative position help in the identification of compound of the mixture.
12. The Rf value is calculated using the formula:
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Rf = Distance traveled by the solute
Distance traveled by the solvent.
Result: The given mixture contains ______________ and ______________ with an Rf value of
____________ and _____________.
Observation and calculation:
Rf = Distance traveled by the solute
Distance traveled by the solvent.
Sl. no distance traveled by distance traveled by Rf value
the solvent the solute
The given mixture contains ____________ and ______________
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3. SEPARATION OF PHOTOSYNTHETIC PIGMENTS USING THIN LAYER
CHROMATOGRAPHY.
Aim: To separate the various components of photosynthetic pigments using thin layer chromatography.
Principle: A thin layer of the stationary phase is formed on a suitable flat surface such as glass plates.
Since the layer is so thin, the movements of mobile phase across the layer, generally by simple capillary
movements is rapid. As the mobile phase moves across the thin layer from one edge to the other, it
transfers any analyte placed on the layer. The movement of the analyte is expressed by its retardation
factor Rf.
Rf = Distance moved by analyte from origin
Distance moved by solvent from origin
Materials required:
Silica gel: On plates 20 x 20 cm plates. This is prepared by mixing 30 gm of silica gel in 70 ml water.
Photosynthetic pigments: The pigments are obtained by grinding the young leaves in acetone. The solution
is filtered and used as analyte.
Solvent Mixture: 75 parts petroleum ether and 25 parts acetone.
Developing chromatography: Pour the developing solvent into the tank to a depth of 1.5 cm. Allow it to
stand for at least an hour with a cover plate over the top of the tank to ensure that it is saturated.
Preparation of plates: Place dry glass plates on the plastic base plate over a plane surface. Prepare a slurry
of the adsorbent in water, cool and pour the slurry over the glass plate at a thickness of 0.25cm by moving
the applicator at a uniform speed from one end to the other. Heat the plates in an oven at 100° -120° C for 1 –
2 hr to remove the moisture and to activate the adsorbent on the plate.
Procedure:
1. Leave 3 cm distance from one end of the silica glass plate.
2. Apply the sample by means of capillary tubes.
3. Allow the sample to dry and repeat the process two more times
4. place the silica plate vertically in the tank so that it stands in the solvent with the spotted end dipping
in the solvent. Replace the cover plate.
5. Once the solvent reaches three fourth of the plate, remove it from the tank and dry it.
Visualizing: The plates are kept under UV lamp and checked for different spots or the chlorophyll bands can
be directly visualized with the naked eyes.
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Result:
Dark green -chlorophyll a
Light green – chlorophyll b
Yellow- xanthophyll
Orange- carotenoid
Observation and calculation:
Rf = Distance traveled by the solute
Distance traveled by the solvent.
sl. no distance traveled distance traveled Rf value
by the solvent by the solute
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4. Determination of absorption maximum of a solution
Aim: To determine the absorption maxima of the given unknown solutions
Principle: the coloured compounds have their own characteristic absorption spectra and careful
selection of the wavelengths where maximum absorption is found, enables a mixture of two coloured
substances to be analyzed.
Material required:
Colorimeter with a series of filters
Bromophenol blue, methyl orange, saffranin, malachite green
Procedure:
1. The coloured dye is taken in a test tube
2. A blank solution is prepared by taking distilled water without the dye.
3. The colorimeter is set to wavelength 420nm
4. The colorimeter is set the zero absorbance (100% transmittance) with blank.
5. The absorbance is for bromophenol blue at 420 is taken.
6. The instrument is set to 490nm, 540 nm, 590nm and 650nm. The colorimeter is set to zero each
time with blank solution every-time when the absorbance (O.D.) is taken,
7. The absorbance is noted for methyl orange, saffranin, and malachite green at 420nm, 490nm,
540nm, 590nm and 650nm recorded.
8. A standard graph is plotted taking absorbance of each dye on x –axis and O.D. on y-axis.
9. The maximum absorbance for each dye is recorded.
Result:
1. The wavelength that gives maximum absorption for bromophenol blue =_______________
2. The wavelength that gives maximum absorption for methyl orange =__________________
3. The wavelength that gives maximum absorption for saffranin=-------------
4. The wavelength that gives maximum absorption for malachite green =-------------
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Observation:
Wavelength O.D. for O.D. for O.D. for O.D. for
in nm Bromophenol Saffranin Methyl Malachite
blue orange green
420
490
540
590
650
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5. CYCLOSIS IN HYDRILLA
Aim: To demonstrate and study the process of cyclosis in Hydrilla
Introduction:
Living cells perform various kinds of biological movements and they show internal flowing of
cytoplasm called cyclosis or cytoplasmic streaming. cyclosis is found to occur in all kinds of cells. In
many plant cells cytoplasm is seen to flow around the vacuole. In a plant cell cytoplasm forms a thin
layer surrounding the large central vacuole. Continuous current can be seen in the cell which displaces
cytoplasm granules and organelles like plastids. The cytoplasmic streaming may be initiated by
chemicals or by light. plant growth hormones such as auxins increase the rate of cyclosis. Cyclosis is
stopped by mechanical injuries and electrical shocks. Hydrilla is an aquatic flowering plant found in
ponds.
Procedure:
1. A hydrilla plant is taken from an pond in a watch glass containing water.
2. A leaf is removed from a tip of the branch.
3. It is placed on a slide with the ventral side up on a drop of water. A cover slip is placed on it.
4. It is observed under low power and then under high power.
5. study the individual cells to determine any internal movement
Observation:
cells are rectangular in shape and are surrounded by cell wall
there is a central vacuole and the cytoplasm occupies the peripheral position
practically all the cells appear to possess the green oval bodies called chloroplasts
chloroplasts lie quite close to the cell wall and they are constantly in slow movement
nucleus also occupies the peripheral position but is hidden by chloroplasts
Inference:
Rapid movement of chloroplasts in the peripheral cytoplasm is seen in the leaf cells of Hydrilla.
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