Group 1

Group 1
(B.SC. BIOTECHNOLOGY)-
1. AYANAVA CHATTERJEE (30008221001)
2. ASMITA BHATTACHARJEE (30008221002)
3. SREYASHI RAY (30008221004)
4. SUTANUKA DAS (30008221008)
5. SHARMISTHA PAL (30008221011)
6. JAYRAJ BISWAS (30008221012)
7. SUKRISHA KARMAKAR (30008221016)
(B.SC. BIOINFORMATICS)-
8. SAMPRIKTA BERA (30059921004)
9. DEEPSHIKHA DWARI (30059921005
Experiment : Application of UV Visible
Spectrophotometer
Aim
To study the application of a UV visible Spectrophotometer / To determine the

concentration of protein samples using UV Visible spectroscopy at 540 nm
Principle
The Beer-Lambert Law, also known as the Beer-Lambert-Bouguer Law, is a fundamental
principle in the field of spectroscopy, which describes the relationship between the
absorbance of light by a solution and the concentration of the absorbing species in the
solution.
According to the Beer-Lambert Law, the absorbance of a solution is directly proportional to

the concentration of the absorbing species and the path length of the light through the
solution. The law can be expressed mathematically as:
A = εcl
where A is the absorbance of the solution, ε is the molar absorptivity (also known as the
extinction coefficient), c is the concentration of the absorbing species, and l is the path
length of the light through the solution.
Operating Procedure
UV–visible spectrophotometers have five main components: the light source,
Monochromator, Sampleholder, Detector, and Interpreter.
1. The light source- Tungsten filament lamps and Hydrogen- Deuterium lamps
are the mostwidely used. For the source of visible light we use tungsten lamp
(400-700nm) and for the source of UV light we use D 2 or deuterium lamp (200-
400nm).
2. Monochromator- Monochromators generally are composed of prisms and slits.

Most of the spectrophotometers are double beam spectrophotometers.The
radiation emitted from the primary source is dispersed with the help of rotating
prisms.The various wavelengths of the light source which are separated by the
prism are then selected by the slits such the rotation ofthe prism results in a series
of continuously increasing wavelengths to pass through the slits forrecording
purposes. The beam selected by the slit is monochromatic. The beam selected by
theslit is monochromatic and further divided into two beams with the help of
another prism. (Onlyfor the double beam UV spectrometer)
3. Sample holder- The monochromatic light passes through the sample which
contain referencecells in a quartz cuvette because glass will absorb all of the light
higher than 300nm. So a quartzcuvette will be more practical as the absorbance
cut-off is about 160nm. One of the two dividedbeams is passed through the sample
solution and the second beam is pass through the reference solution. (Only for
double beam)
4. Detector- The detector is a in-line device that measures the UV absorbance of

the sample and provides a continuous signal that can be used to quantify the
amount of the chromophoriccompounds emerging from the sample.
a. The photomultiplier tube is the most popular detector used in UV- Visible
spectroscopy. It comprises of a photosensitive cathode, anode and several dynodes.
Photons entering the tube strike the cathode resulting in emission of electrons.
b. There are two different detectors for two different sample. One of the
photocells receives the beam from the sample cell and the second detector
receives the beam from the reference. (Only for double beam spectrometer)
5. Interpreter- It’s a device that interpret the signal that received from sample
through detector.The computer stores all the data generated and produces the
spectrum of the desired compound.
FIG: Single beam & Double beam Spectrophotometers

Application
How to estimate protein assay by Biuret method
- Preparation of Biuret Reagent:
3gm of CuSo4 and 9m of Na-k tartrate is dissolved in 500 ml of 0.2mol /L NaoH. 5gm
of KIsolution is made up to 1 litre with 0.2 mol/L NaoH.
- Protein Estimation:
1. First, five dry and clean test tubes are taken and marked
respectively with apermanent marker.
2. Preparation of Protein Concentration by serial dilution is made
CONCENTRATION BSA STOCK H2O FINAL VOLUME

( mg/ml)
Blank 0 1 1ml
1 100 ml 900 1ml
2 200 ml 800 1ml
4 400 ml 600 1ml
8 800 ml 200 1ml
3. Now with each test tubes 1ml protein + 4ml Biuret Reagent is added.
4. The samples are incubated at room temperature for 5minutes.
5. Finally the Absorbance of the five samples are checked at 540 nm using
UV visibleSpectrophotometer.
Observation
CONCENTRATION BSA STOCK H2O FINAL VOLUME
(mg/ml)
1 50 ml 450 ml 0.5
2 100 ml 400 ml 0.5
4 200 ml 800 ml 0.5
8 400 ml 1000ml 0.5
Precautions
1.Take out the desiccant in the sample chamber before starting the machine. It is
forbiddento open the sample chamber cover during the self-checking process of
the instrument.
2. The solution in the cuvette should be 2/3~4/5 of the height of the cuvette and
it should not be too full to prevent the liquid from overflowing and corroding the
instrument. Keep the cuvette clean during the measurement and wipe the liquid
droplets on the wall with lens cleaning paper. Do not pinch the translucent
surface with your hands. While measuringwavelength quartz cuvette is required.
3. During the measurement it is forbidden to put reagents or liquid substance

on the surface of the instrument. If the solution has overflow or other reasons
the sample tankshould be cleaned up as soon as possible.
4. After performing the experiment pour out the solution in the cuvette then rinse
the cuvette with distilled water or organic solvent until it is clean and dry it upside
down. Turn off the power, put the desiccant into the sample chamber, cover the
dust cover and registerfor use.
5. If the absorption value is abnormal, double-check in turn whether wavelength

setting is correct or whether the zero is adjusted during measurement or whether
the cuvette is usedcorrectly.

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Group 1

1. AYANAVA CHATTERJEE (30008221001)

2. ASMITA BHATTACHARJEE (30008221002)

3. SREYASHI RAY (30008221004)

4. SUTANUKA DAS (30008221008)

5. SHARMISTHA PAL (30008221011)

6. JAYRAJ BISWAS (30008221012)

7. SUKRISHA KARMAKAR (30008221016)

8. SAMPRIKTA BERA (30059921004)

9. DEEPSHIKHA DWARI (30059921005

Experiment : Application of UV Visible

To study the application of a UV visible Spectrophotometer / To determine the

According to the Beer-Lambert Law, the absorbance of a solution is directly proportional to

2. Monochromator- Monochromators generally are composed of prisms and slits.

4. Detector- The detector is a in-line device that measures the UV absorbance of

FIG: Single beam & Double beam Spectrophotometers

- Preparation of Biuret Reagent:

CONCENTRATION BSA STOCK H2O FINAL VOLUME

3. During the measurement it is forbidden to put reagents or liquid substance

5. If the absorption value is abnormal, double-check in turn whether wavelength

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