BT510 Analytical

Biotechnology Lab record

M.TECH BT
IITG

Table of Contents
Experiment 1 Visible spectroscopy: Protein estimation by Bradford
reagent
3
Experiment 2 Fluorescence Spectroscopy 1

Experiment 3 Fluorescence Spectroscopy 2

12

Experiment 4 Stock solution preparation

15

Experiment 5 Thin Layer Chromatography for separation of amino acids

17
Experiment 6 High Performance Liquid Chromatography 23
Experiment 7 Field emission Scanning electron microscopy

26

Experiment 8 Fluorescence Microscopy: viewing cells stained with

fluorescent dyes 29

Experiment-1
Aim: Use Bradford assay to estimate the protein concentration of following:
a) Unknown Protein sample
b) Milk sample
Theory/Principle: Bradford assay is based on the UV-Vis absorption spectroscopy
which involves ultraviolet and visible radiation interacting with matter. This
interaction causes electronic transitions (promotion of electrons from the ground
state to a high energy state). Absorption of light corresponds to the concentration of
the solute being measured which is as per Beers law.
Bradford assay is a dye based assay involving binding of Coomassie Blue dye to
proteins.

Figure 1: Structure of Coomassie Blue

Free Coomassie Blue G250 can exist in four different ionization states with pK1,
pK2, and pK3 of 1.15, 1.82, and 12.4. At pH 0, both the sulfate groups are
negatively charged and all three nitrogens are positively charged giving the dye +1
net charge (the red form of the dye). Around pH 1.5, the neutral green form of the
dye predominates. At neutral pH, the dye has a net charge of +1 (the blue form of
the dye). The red, green, and blue forms of the dye absorb visible radiation with
absorption maxima at 470, 650, and 590 nm, respectively. It is the anionic form of
the dye that binds to the protein. Binding of the blue form of Coomassie Blue G250
with proteins causes red-shift in its absorption spectrum; the absorption maximum
shifts from 465 to 595 nm. The dye binds more readily to the cationic residues,
lysine and arginine. The interaction strength and the staining intensity depend on
the specific protein.
Advantages of Bradford assay:
1)
2)
3)
4)

Is faster and involves fewer mixing steps.

Does not require heating.
Gives a more stable colorimetric response.
The sensitivity is within 10-100 g but with micro assays as little as 2 g
protein can be detected.

Disadvantages of Bradford assay:

1) Prone to influence of non-protein sources e.g. detergents.

2) Response is dependent on amino acid composition of the protein

3) Becomes progressively non-linear at the high end the of its protein
concentration range.
4) Causes blue staining of cuvettes.

Materials required:
i)

ii)

Equipments
Spectrophotometer
Glass or polystyrene cuvettes
Opaque eppendorfs
Chemicals/reagents
Bradford reagent (Dissolve 100 mg Coomassie Brilliant Blue G-250
in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute
to 1 litre when the dye has completely dissolved, and filter through
Whatman paper just before use.)
Bovine serum albumin(BSA)
Distilled water
Unknown sample
Milk sample

Procedure:
1) A set of standards containing 5, 7.5, 10, 12.5, 15 and 20 l of Bovine serum
albumin (BSA 2.0mg/ml stock in distilled water) in separate tubes was
prepared. Distilled water was then added to each tube to bring the volume to
100 l.
2) After preparing appropriate dilutions Bradford reagent was added and the
eppendorfs were incubated for 5mins.
3) The unknown protein sample was also diluted appropriately and incubated
with Bradford solution.
4) Absorbance was then measured at 595nm.
5) Above procedure was followed for milk sample as well.

Observations:
a) For unknown protein sample:
Protein
S no.

Distilled water
(l)

Final protein
conc. (g/ml)

Bradford
reagent (ml)

A595 nm

(l)

(g)

100

1.0

10

95

100

1.0

0.198

7.5

15

92.5

150

1.0

0.339

10

20

90

200

1.0

0.524

12.5

25

87.5

250

1.0

0.621

15

30

85

300

1.0

0.638

20

40

80

400

1.0

0.745

Unknown
Sample (100
l)

1.0

0.321

Concentration vs Absorbance
0.8

f(x) = 0x + 0.04
R = 0.94

0.7
0.6
0.5

Absorbance at 595nm

0.4
0.3
0.2
0.1
0
0

50

100

150

200

250

300

350

400

450

Concentration (g/ml)

Graph 1- Standard curve of absorbance of BSA at 595nm for estimating

protein concentration in unknown sample
b) For milk sample
Protein
S no.

Distilled water
(l)

Final protein
conc. (g/ml)

Bradford
reagent (ml)

A595 nm

(l)

(g)

100

1.0

10

95

100

1.0

0.3206

7.5

15

92.5

150

1.0

0.509

10

20

90

200

1.0

0.732

12.5

25

87.5

250

1.0

0.9521

15

30

85

300

1.0

1.292

20

40

80

400

1.0

1.5053

Unknown
Sample (100
l)

1.0

1.1828

Concentration vs Absorbance
1.6
f(x) = 0x - 0.04
R = 0.98

1.4
1.2
1

Absorbance at 595nm

0.8
0.6
0.4
0.2
0
0

50

100

150

200

250

300

350

400

450

Concentration (g/ml)

Graph 2- Standard curve of absorbance of BSA at 595nm for estimating

protein concentration in milk sample

Calculations:
a) For unknown protein sampleThe equation for the standard curve is obtained as y=0.004x -0.042
Where y is the absorbance, m is the slope of the curve, x is the concentration
of protein and c is y-intercept.
On substituting the value of absorbance for the unknown sample in the above
equation we obtain the concentration of protein as:
0.321=0.002x + 0.0434
0.002x=0.321-0.0434
x=138.8 g/ml= 138.8 mg/l= 0.139 g/l

b) For milk sampleThe equation for the standard curve is obtained as y = 0.004x - 0.042
On substituting the value of absorbance for the milk sample in the above
equation we obtain the concentration of protein as:
1.1828=0.004x - 0.042
0.004x=1.1828+0.042
x=306.2 g/ml = 306.2 mg/l=0.306 g/l

Result: The concentration of unknown protein sample was found to be 0.14 g/l and
that of milk sample to be 0.306 g/l.

EXPERIMENT No. 2
TITLE:
Equilibrium unfolding of bovine serum albumin monitored using tryptophan
fluorescence.

OBJECTIVE:
To study the effect of quencher (water) on tryptophan present in given protein at
different temperature using fluorescence.

PRINCIPLE:
The folded structure of a protein is crucial for its function. The factors that govern
protein folding are manifold. They include the protein sequence and other external
parameters like solvent polarity, temperature, pH and salt concentration. It is still
not possible to unambiguously predict the 3 dimensional structure of a protein from
its primary sequence.
Here, we monitor the equilibrium unfolding of bovine serum albumin by monitoring
the fluorescence intensity of the Trp in the protein. Here, we are considering
temperature as a means to unfold the protein structure. So, as we will increase the
temperature, incrementally there would be exposure of the Trp from core of the
protein to the outside. Hence, increase in the quenching. The Trp fluorescence
intensity and emission wavelength serve as probes for the environment surrounding
the Trp residue in the protein.

METHODOLOGY
REQUIREMENTS:
A) Buffer

Phosphate buffered saline (PBS), pH 7

B) Protein stock solution

Bovine serum albumin (BSA) at 12mg/ml is prepared in Phosphate buffered

saline (PBS), pH 7.

C) Glasswares/ plasticwares

Eppendorfs (1.5ml)- 5 Nos.

Micropipettes(P1000, P20)
Microtips (1000l, 20l)
Fluorescence cuvette

D) Equipments

Vortex Mixer

Spectrofluorimeter

PROCEDURE:
1. Aliquot buffer and protein in Eppendorf tubes as mentioned below.
Samples

PBS (ml)

BSA Stock solution (l)

Blank
Sample 1
Sample 2
Sample 3
Sample 4

1.0
0.992
0.992
0.992
0.992

8.3
8.3
8.3
8.3

Incubation
Temperature (C)
Room temperature
Room temperature
50
70
90

2. Measure 0.992 ml of PBS and 8.3 l of BSA. Pipette it in Eppendorf tube and
incubate it at room temperature. Similarly, sample 2, 3 and 4 are also
incubated at 50C, 70C and 90C respectively.
3. Incubate all samples for 15 minutes at their respective temperatures.
Setting up the spectrofluorimeter:
Experiment shall be performed on a spectrofluorimeter. Set the excitation
wavelength at 295nm and the emission wavelength range from 320 to 380nm. Set
the excitation bandwidth at 2nm and emission bandwidth at 5nm.

OBSERVATIONS:
1. Measure fluorescence emission spectrum of blank 1.
2. Under same conditions measure the emission spectrum of sample 1-4.
3. Store results.

CALCULATIONS:
1. Subtract the blank 1 spectrum from sample 1 spectrum to correct for Raman
Scatter. Repeat this vice versa for Sample 2. Sample 3 and Sample 4 spectra
also.
2. Plot a graph between Emission wavelength vs Fluorescence Intensity.
3. Plot a Graph between temperature vs Fluorescence Intensity at 355nm.

RESULTS:
Following graphs were obtained.
Graph-1: Emission wavelength vs Fluorescence Intensity at different temperatures.

Emission wavelength vs Fluorescence Intensity

600000
500000

Flourescence Intensity

400000

Intensity at Room
temperature

300000

Intensity at 50 C

200000

Intensity at 70 C
Intensity at 90 C

100000
0
320 330 340 350 360 370 380
Emission Wavelength (nm)

Graph-2: Temperature vs Fluorescence Intensity at 355nm.

Decrease in Fluorescence intensity wrt temp.

600000
500000
400000
Fluorescence Intensity

300000

Data taken at 355nm

200000
100000
0
20

40

60

80

Temperature(Celcius)

100

DISCUSSION: The fluorescence intensity was found to decrease with increase in

temperature due to protein molecules getting denatured (unfolded) and tryptophan
indole ring getting exposed to water molecules which act as quencher of
fluorescence. This quenching results in hypochromic shift in the fluorescent
intensity.

EXPERIMENT NO. 3
TITLE:
Equilibrium unfolding of bovine serum albumin monitored using tryptophan
fluorescence.

OBJECTIVE:
To study the effect of quencher (water) on tryptophan present in given protein at
different molar concentration of urea using fluorescence.

PRINCIPLE:
The folded structure of a protein is crucial for its function. The factors that govern
protein folding are manifold. They include the protein sequence and other external
parameters like solvent polarity, temperature, pH and salt concentration. It is still
not possible to unambiguously predict the 3 dimensional structure of a protein from
its primary sequence.
Here, we monitor the equilibrium unfolding of bovine serum albumin by monitoring
the fluorescence intensity of the Trp in the protein. Here, we are considering
temperature as a means to unfold the protein structure. So, as we will increase the
temperature, incrementally there would be exposure of the Trp from core of the
protein to the outside. Hence, increase in the quenching. The Trp fluorescence
intensity and emission wavelength serve as probes for the environment surrounding
the Trp residue in the protein.

METHODOLOGY
REQUIREMENTS:
A) Buffer

Phosphate buffered saline (PBS), pH 7

B) Protein stock solution

Bovine serum albumin (BSA) at 12mg/ml is prepared in Phosphate buffered

saline (PBS), pH 7.

C) Glasswares/ plasticwares

Eppendorfs (1.5ml)- 5 Nos.

Micropipettes(P1000, P20)
Microtips (1000l, 20l)
Fluorescence cuvette

D) Equipments

Vortex Mixer
Spectrofluorimeter

PROCEDURE:
1. Aliquot buffer and protein in Eppendorf tubes as mentioned below.
Buffer
S No.

Sample
Name

Name

Quantit
y

Blank 1

NaH2PO4 buffer

BSA
Quantity from BSA stock (0.5
mg/ml)(ml)
.

2
3
4
5
6
7
8
9
10

Sample 1
Blank 2
Sample 2
Blank 3
Sample 3
Blank 4
Sample 4
Blank 5
Sample 5

NaH2PO4 buffer
2 M Urea buffer
2 M Urea buffer
4 M Urea buffer
4 M Urea buffer
6 M Urea buffer
6 M Urea buffer
8 M Urea buffer
8 M Urea buffer

0.992
1
0.992
1
0.992
1
0.992
1
0.992

8.3 l
.
8.3 l
.
8.3 l
.
8.3 l
.
8.3 l

2. Measure 0.992 ml of PBS and 8.3 l of BSA. Pipette it in Eppendorf tube and
incubate it at room temperature. Similarly, sample 2, 3 and 4 are also
incubated at 50C, 70C and 90C respectively.
3. Incubate all samples for 15 minutes at their respective temperatures.
Setting up the Spectrofluorimeter:
Experiment shall be performed on a Spectrofluorimeter. Set the excitation
wavelength at 295nm and the emission wavelength range from 320 to 380nm. Set
the excitation bandwidth at 2nm and emission bandwidth at 5nm.

OBSERVATIONS:
1.
2.
3.
4.

Measure fluorescence emission spectrum of blank 1.

Under same conditions measure the emission spectrum of sample 1-4.
Store results.
Repeat steps 1 to 3 with blanks and samples for 2M , 4 M, 6 M and 8 M Urea
concentrations.

CALCULATIONS:
1. Subtract the blank 1 spectrum from sample 1 spectrum to correct for Raman
Scatter. Repeat this vice versa for Sample 2. Sample 3 and Sample 4 spectra
also.
2. Plot a graph between Emission wavelength vs Fluorescence Intensity.
3. Plot a Graph between [Urea] vs Fluorescence Intensity at 355nm.

RESULTS:
Graph1: Emission wavelength vs Fluorescence intensity with different
concentrations of Urea

Emission wavelength vs Fluoresence intensity

600000
500000

Fluoresence intensity

400000

NaH2PO4 Buffer

300000

4M Urea

200000
100000
0
300 320 340 360 380 400

Wavelength (in nm)

Graph 2: Concentration of Urea vs Fluorescence intensity at 355 nm

2M Urea
6M Urea
8M Urea

Urea conc. vs Fluorescence intensity

600000
500000
400000

Fluorescence intensity(at 355nm) 300000

200000
100000
0
0

Urea conc.(in M)

DISCUSSION: The fluorescence intensity was found to decrease with the increase
in concentration of urea due to protein molecules getting denatured (unfolded) and
tryptophan indole ring getting exposed to water molecules which act as quencher of
fluorescence. This quenching results in hypochromic shift in the fluorescent
intensity.

EXPERIMENT No. 4
Aim: Preparation of stock solutions of EDTA, Sodium Hydroxide and TAE buffer
1) Prepare a stock solution of 0.5M Sodium hydroxide(NaOH)
2) Prepare a stock solution of 0.5M EDTA in 50ml.
3) TAE buffer- 50X stock solution in 100ml

Theory:
Buffer solutions
Buffer solutions are solutions which resist change in hydronium ion and the
hydroxide ion concentration (and consequently pH) upon addition of small amounts
of acid or base, or upon dilution. Buffer solutions consist of a weak acid and its
conjugate base (more common) or a weak base and its conjugate acid (less
common). The resistive action is the result of the equilibrium between the weak acid
(HA) and its conjugate base (A):
HA(aq) + H2O(l) H3O+(aq) + A(aq)
Any alkali added to the solution is consumed by hydronium ions. These ions are
mostly regenerated as the equilibrium moves to the right and some of the acid
dissociates into hydronium ions and the conjugate base. If a strong acid is added,
the conjugate base is protonated, and the pH is almost entirely restored. This is an
example of Le Chatelier's principle and the common ion effect. This contrasts with
solutions of strong acids or strong bases, where any additional strong acid or base
can greatly change the pH.
Chemical Equilibrium
Chemical equilibrium is the condition which occurs when the concentration of
reactants and products participating in a chemical reaction exhibit no net change
over time. Chemical equilibrium may also be called a "steady state reaction." This
does not mean the chemical reaction has necessarily stopped occurring, but that
the consumption and formation of substances has reached a balanced condition.
The quantities of reactants and products have achieved a constant ratio, but they
are almost never equal. There may be much more product or much more reactant.
Buffering Capacity
Buffer capacity is a measure of the efficiency of a buffer in resisting changes in pH.
Conventionally, the buffer capacity is expressed as the amount of strong acid or
base, in gram-equivalents, that must be added to 1 liter of the solution to change its
pH by one unit.
TAE Buffer
TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and
EDTA. The useful buffer range for tris (7-9) coincides with the physiological pH
typical of most living organisms. It is historically the most common buffer used for

agarose gel electrophoresis in the analyses of DNA products resulting from PCR
amplification, DNA purification protocols or DNA cloning experiments. This buffer
has a low ionic strength and low buffering capacity. EDTA acts as chelating agent
and chelates bivalent ions such as those of Magnesium, rendering them unavailable
for DNase which keeps the DNA from degrading.

Procedure:
1) For 0.5M NaOH solution in 50ml
Dissolve 1g of NaOH in 50ml of water.
2) For 0.5M EDTA in 50ml
EDTA will not go completely into solution until the pH is adjusted to about 8.0.
For a 50ml stock solution of 0.5 M EDTA, 9.305 grams of EDTA disodium salt
(MW = 372.2) is taken and dissolved in water and the pH is adjusted with
sodium hydroxide (NaOH).
3) For 50X TAE buffer
Tris Base- 24.2% w/v is taken which equates to 12.1g.
Glacial Acetic Acid- 5.71% v/v is taken which equates to 2.855ml
0.5M EDTA (pH 8.0) 10% v/v is taken which equates to 5ml
Tris base 12.1g is dissolved in 30ml water and then glacial acetic acid
2.855ml and 5ml of EDTA solution are added to it. The volume is then made
upto 50ml with water.

Result: The stock and buffer solutions were prepared and stored for future use.

Experiment-5
Aim: To separate different amino acids present in unknown sample using thin layer
chromatography.
Principle:
The thin layer chromatography technique is a method for analyzing mixtures by
separating the compounds in the mixture. TLC can be used to help determine the
number of components in a mixture, the identity of compounds, and the purity of a
compound. In this method, the silica or alumina as a stationery phase is coated on
to a glass or aluminum foil as thin layer and then a sample is allowed to run in the
presence of a mobile phase (solvent). In comparison to other chromatography
techniques, the mobile phase runs from bottom to top by diffusion (in most of the
chromatography techniques, mobile phase runs from top to bottom by gravity or
pump). As sample runs along with the mobile phase, it gets distributed into the
solvent phase and stationery phase. The interaction of sample with the stationery
phase retard the movement of the molecule whereas mobile phase implies an
effective force onto the sample. Suppose the force caused by mobile phase is F m
and the retardation force by stationery phase is F s, then effective force on the
molecule will be (Fm-Fs) through which it will move. The molecule immobilizes on the
silica gel (where, Fm=Fs) and the position will be controlled by multiple factors.
Ninhydrin is used in amino acid analysis of proteins. Most of the amino acids, except
proline, are hydrolyzed and react with ninhydrin.

1. Nature or functional group present on the molecule or analyte.

2. Nature or composition of the mobile phase
3. Thickness of the stationery phase.
4. Functional group present on stationery phase.
If the distance travelled by a molecule on TLC plate is D m whereas the distance
travelled by the solvent is Ds, then the retardation factor (Rf) of molecule is given
by:

Rf value is characteristic to the molecule as long as the solvent system and TLC
plate remains unchanged. It can be used to identify the substance in a crude
mixture.

Rf = Y/X (always 1)

More polar component will make bond to the silica more strongly than less polar
component. Hence, less polar component has travelled the farthest whereas more
polar solvent travel the least.
The Rf (retardation factor) depends on the following parameters:

solvent system

absorbent (grain size, water content, thickness)

amount of material spotted

temperature

Because all those variables are difficult to keep constant, a reference compound is
usually applied to the plate as well. These effects normally cause an increase in Rf
values. However, in the case of layer thickness, the Rf value would decrease
because the mobile phase moves slower up the plate.
Instruments, chemicals and glassware:
Material Required

1. Eluent. [n-butanol, acetic acid (purity 98 100 %) and distilled water in volume ratio
5:1:5.]
2. Ninhydrin Solution. [0.3 g of ninhydrin in 100 ml n-butanol. Add 3 ml of glacial acetic
acid.]
0.02 M solutions of amino acids (leucine, methionine, alanine, serine and mix).
3. TLC Silica gel 60 F254 plate.
4. Chromatographic chamber.
5. Glass capillaries for spotting the samples.
6. Graduated test-tube.
7. Graphite pencil.
8. Solution of ninhydrin.
9. Ruler.
10.
Scissors.
11.
Rubber gloves.
Analytical procedure

1. Gloves must be used during this work to avoid contamination of TLC Silica gel
60 F254 plate with amino acids from skin, and for protecting skin from
solvents and ninhydrin.

2. Mark the starting line on the TLC Silica gel 60 F254 plate approximately 0.5
cm from the edge with graphite pencil. Also mark the locations where the
samples will be spotted. The neighboring spots should be about 5 mm apart
from each other and at least 5 mm away from the papers edge.

3. The spots of individual amino acids and sample solution are applied to the
TLC Silica gel 60 F254 plate. Use separate clean and dry glass capillary for
each solution. Dip the capillary into solution some solution is drawn into the
capillary. With the filled capillary touch the prepared location on TLC Silica gel
60 F254 plate.

4. The spot on the TLC Silica gel 60 F254 plate should not be bigger than 2-3
mm.

5. After application of samples let the spots dry. To start the analysis, insert the
TLC Silica gel 60 F254 plate into Chromatographic chamber such a way that
spot should not be below the solvent level.

6. Elution is stopped when the solvent front has traveled up the plate until 7-10
mm from the top.

7. Remove the plate from Chromatographic chamber and place it on a sheet of

filter paper. After 2-3 minutes mark the eluent front with pencil and dry the
paper. When the plate is dry, take it into the fume hood and spray it with
solution of ninhydrin until the paper is slightly damp.

8. Chromatographic plate should lie at 45 angle while spraying. The

chromatographic paper is again put for drying for 15 min.

9. Draw the contours and centers of the chromatographic bands.

Observation:
Following results were obtained after ninhydrin was sprayed onto TLC column.

Figure.1- TLC Plate

Calculations:
Distance travelled by the solvent= 4.5 cm

Distance travelled by each of the known samples:

1.
2.
3.
4.

Methionine= 1.8 cm
Alanine= 0.7 cm
Serine= 0.6 cm
Leucine= 2.2 cm

We know,

Therefore, Rf of:

1. Methionine = 1.8/4.5 = 0.4

2. Alanine= 0.7/4.5 = 0.16
3. Serine = 0.6/ 4.5= 0.13
4. Leucine =2.2/4.5 = 0.49
Result:
From the observation we can infer that the mix sample is having 3 spots with
distance travelled 2.2 cm, 1.5 cm, 0.6 cm respectively indicating sample leucine,
methionine and alanine or serine in the mixture.

Experiment-6
Aim: To perform High Performance Liquid Chromatography (HPLC) on a sample
containing
Gramicidin.
Principle:
The principle on which chromatographic methods are based is simple. Compounds
in a mixture are separated from each other based on their preferences for one of
two different solvents (or phases) in contact with each other. For example if a polar
solvent and a nonpolar solvent are brought into contact, polar molecules will prefer
to be in the polar solvent, while nonpolar molecules prefer to be in the nonpolar
solvent. In a chromatography experiment one of these solvents is stationary
(the stationary phase), while the other solvent (the mobile phase) flows over it.
HPLC utilizes a small-diameter column packed with small particles coated with the
stationary phase.
A piston-based pump continuously delivers liquid mobile phase at a steady flow rate
from the mobile phase reservoir through the system. A very small volume of
sample is injected with a syringe into the flowing mobile phase where it is carried to
the column. In the column, the various components of the sample mixture are
separated from each other into bands. These bands elute from the column and
move toward the detector. Several different types of HPLC detectors are available;
we will use a UV-vis spectrophotometric detector that measures the absorbance of
the flowing stream of liquid. When a species with an absorbance different from the
mobile phase passes through the detector, a peak is generated. The result is a plot
of detector signal (absorbance) vs. time. This plot is called a chromatogram.
The chromatographic column you will use in this experiment is a reversedphase column, meaning that the stationary phase is nonpolar (hydrophobic). The
column consists of small, porous silica particles that have hydrocarbon chains
eighteen carbon atoms long (a C-18 column) chemically bonded to their surfaces.
Mobile phases used in reversed-phase columns are polar such as mixtures of water
and methanol or acetonitrile. In this experiment you will use a mobile phase which
is a mixture of 100% Acetonitrile and 10% Acetonitrile. Separation of components in
a sample relies on different substances having different preferences for the mobile
and stationary phases. Molecules that are very polar will elute from the column
early, since they prefer to stay in the mobile phase. Nonpolar molecules, preferring
the environment of the stationary phase, will be retained on the column for longer
periods of time.

For our experiments we use a Shimadzu HPLC system. There are pumps and mobile
phase containers. This allows us to pump the buffer from one pump and the

methanol from the other. The computer software will allow us to mix the two liquids
in our desired 80:20 ratio. If this ratio stays constant throughout the experiment,
we are performing an isocratic separation. Sometimes, we can improve a
separation by changing the ratio of mobile phases over the course of the
experiment. This is referred to as gradient elution.

Schematic diagram of HPLC

Chemicals used:

Gramicidin

HPLC-grade Acetonitrile

Milli-Q Water

DMSO

Procedure:
Preparation of HPLC Mobile Phase -

1.

The mobile phase we will use is 100% Acetonitrile and 10 % Acetonitrile. Vacuum
filter the solution through a 0.45-mm nylon filter membrane.

2.

Pour the solution into the Mobile Phase A container and replace the cap. Make
sure the delivery tube connecting it to Pump 1 is firmly inserted all the way to the
bottom.

3.

Check to see that the HPLC-grade Acetonitrile container (Mobile Phase B) is

adequately full. Make sure that the delivery tube connecting it to Pump 2 is firmly
inserted all the way to the bottom.

4.

The software will be set to mix them in a 80:20 ratio.

Preparation of Gramicidin Standard -

5.

Accurately weigh ~0.3 g into a eppendorf vial and dilute to 0.5 ml with DMSO
and mix.

6.

The sample must then be filtered through a 0.45-mm syringe filter to remove
any small particles that may clog the column. Pour the sample into a clean
disposable syringe equipped with an unused syringe filter. Use the plunger to push
the sample slowly through the filter into a labeled autosampler vial. Cap the vial
with a septum.
Sample is placed in autosampler.
samples in a row automatically.

Observation:

The autosampler will allow us to run many

Data Analysis:
As seen from the obtained chromatogram peaks are obtained at 5.397 mins for
DMSO and at 8.758 mins for Gramicidin.

Experiment-7
Aim: To observe the working of field emission scanning electron microscopy.
Principle: Field emission Scanning electron microscope uses a focused beam of
high energy electrons to generate a variety of signals at the surface of solid
specimens. The resolution of FESEM is about 1nm. The signals from electron-sample
interactions reveal information about the sample including morphology, chemical
composition, crystalline structure and orientation on the surface of the molecule.
The specimen usually requires a vacuum chamber so that even small particle could
deflect the electrons. Electrons are liberated from a field emission source and
accelerated in a high electrical field gradient. Within the high vacuum column these
so-called primary electrons are focussed and deflected by electronic lenses to
produce a narrow scan beam that bombards the object. As a result secondary
electrons are emitted from each spot on the object. The angle and velocity of these
secondary electrons relates to the surface structure of the object. A detector
catches the secondary electrons and produces an electronic signal. This signal is
amplified and transformed to a video scan-image that can be seen on a monitor or
to a digital image that can be saved and processed further.
Instrumentation:
The essential components in the FESEM include:
Electron source (Electron gun)
manipulation system

Electron

Sample stage

Scanning system

Vacuum system

Detector

beam

Electron gun generates free electrons and accelerates these electrons in the range
of 1-40keV. These are called primary scanning electrons. Electron manipulation
system consists of electromagnetic lenses and scanning coils to produce focused
electron beam.

Schematic diagram of Field emission scanning electron microscope

Scanning system involves electron beam interaction with specimen to produce
secondary electrons and backscattered electrons which is collected by a detector.
The signals collected are processed and allowed to visualize as an image using
cathode ray tube and recorded.
Working:
Sample preparation:
Living specimens being prepared for scanning electron microscopy first need to be
killed and fixed. This is usually done using a chemical fixative, such as
glutaraldehyde and osmium. Following fixation, water is chemically extracted from
the specimen using a graded series of ethanol for dehydration. Drying of specimens
is done by critical point drying technique. The dried specimens are mounted on
stubs, mostly double stick, electrically conductive tape and specimen has to be
coated with electrically conductive material (mostly gold is preferred). Samples
prepared for elemental analysis are typically carbon coated by flash evaporation.
Scanning electron microscopy:
The beam of electron is produced from the cathode filament and accelerates in the
range of 0.1 to 40 KV down the sample. Electron beams are controlled by magnetic
lenses (magnetic field). Most SEM use several electromagnetic lenses to reduce the
size of beam. Such lenses are called condenser lenses. By these lenses, the electron
beam focuses on the sample. The primary electrons enter the specimen. The hitting
of primary electron on the specimen results in the formation of different scattering
events. Most important scattering events are
Backscattered electrons: Primary electron escape from the specimen but does
not go through the specimen. These are original beam electrons that have high
energy. It I used to determine the crystal lattices and orientation.

Secondary electrons: Generated when a primary electron dislodges a specimen

electron from the specimen surface and imaging in the secondary ode is that the
contrast and soft shadows of image closely that of specimen illuminated with light.
The imaging process occurs across the specimen which gives the morphology and
topography of samples.
Transmitted electrons: If the specimen is thin enough, primary electrons may
pass through the specimen. These electrons are known as transmitted electrons and
they provide atomic density information. The atomic density information is
displayed as a shadow.
A secondary electron detector magnetically attracts emitted secondary electrons
and also attracted to the scintillator which cause the photon to emit. The emitted
photons from the scintillator reach the photomultiplier tube that amplifies the
signal. The secondary electrons generated directly proportional to the area of the
emitting surface. These signals can be converted into an image by cathode ray tube
(CRT). SEM can also coupled with Energy dispersive X-ray spectroscopy (EDS)to
analyze the distribution of elements within samples.

Applications:

Generate high resolution three dimensional image to represent the morphology

To Analyze elemental distribution using EDS
To characterize the solid materials and to detect and analyze surface fractures,
To Examine surface contaminations,
To identify crystalline structures.

Disadvantages:

SEMs are expensive, large and must be housed in an area free of any possible
electric, magnetic or vibration interference
Special training is required to operate an SEM as well as prepare samples
SEMs are limited to solid, inorganic samples small enough to fit inside the
vacuum chamber that can handle moderate vacuum pressure

Inference:
The working of Field emission scanning electron microscope demonstrated and the
image generation was observed.

Experiment-8
Aim: To observe the working of a fluorescence microscope and viewing cells stained
with fluorescent dyes.
Theory:
Fluorescence is the phenomenon that occurs when a substance absorbs
electromagnetic radiation of a certain wavelength, reaches an excited state and
after a few nanoseconds emits a radiation of a wavelength longer than the absorbed
wavelength. This difference occurs due to non-radiative loss of energy and is known
as Stokes shift. Conventional bright-field microscopy visualises structures of cells
and tissues via the absorption and diffraction of light. In contrast, in FM,
fluorophores become independent light sources when irradiated with the
appropriate exciting wavelengths, while the non-fluorescent surroundings remain
dark. FM takes advantage of (i) Stoke's law, i.e., the difference of wavelength
between exciting and emitted light, and (ii) the high visibility of fluorescent objects
in the dark.
Primary fluorescence: A number of substances such as oils, optical brighteners,
plastic materials and biological materials such as chlorophyll, flavins, and bone
intrinsically emit fluorescent light when irradiated with short wave radiation. This
kind of fluorescence is known as primary fluorescence or inherent autofluorescence.
Secondary fluorescence: Light emission caused by fluorophores added to the
specimen is called secondary fluorescence.
Components of a fluorescence microscope:
Light Source: Fluorescence microscopes require a light source, which illuminates
the field of view
(i) evenly and uniformly (ii) at the exciting wavelength
dictated by the fluorophore and (iii) with high intensity, as the fluorescence yield
amounts only to a few percent of the excitation energy. High-pressure gas discharge
lamps such as mercury (HBO) or xenon (XBO) arcs are most suitable.
Optical Filters and dichroic mirrors: Filters for FM are designed to selectively
transmit light matching the excitation wavelength of a given fluorophore (exciter,
excitation filter), to transmit only the light emitted by fluorophore fluorescence
(emitter, emitter barrier filter), or to separate the exciting and emitted light
(dichroic mirror). Exciter and emitter filter have to be oriented exactly at 45~ to the
dichroic mirror and the emitter filter perpendicular to the optical axis.
Objective lens: It acts as both a condenser for focussing the exciting light and also
collects the emitted fluorescent light. Since the objective lens acts as both
condenser and objective in episcopic fluorescence, quartz objective lenses are
required for deep ultraviolet excitation <= 320 nm. However, sufficient UV above
320 nm passes through fluorite lenses to provide adequate fluorescence. New types
of glass and new lens coatings are leading to better UV transmission in
apochromats.

Recording Systems: With CCD imaging by solid state or video cameras, image
quality and settings can be monitored on screen and adjusted accordingly.

Schematics of an inverted fluorescence microscope

Sample preparation:
Fluorescent dyes are directly taken up by the cells. They are incorporated and
concentrated in specific subcellular compartments. The living cells are then
mounted on a microscope slide and examined under a fluorescence microscope.
Here we are observing MDA-MB breast cancer cell line cells and macrophages.
Observation:

MDM-MB breast cancer cells irradiated with blue light and under green filter

Macrophages irradiated with blue light and under green filter

Inference:
MDA-MB breast cancer cells took up the fluorophore and were consequently
fluorescing with green light while macrophages did not take up the dye and were
not fluorescing. This completed the study of fluorescence microscopy.