ANALYTICAL TECHNIQUES IN

BIOTECHNOLOGY LAB (BM3708)

EXPERIMENT NO: 3

TITLE: UV-Vis spectrophotometry

DEGREE:B.Tech

SUBMITTED BY

GROUP- 1

SUBGROUP-5
SI NO. NAME REGISTRATION NO. SIGNATURE

1 Karthik Peddi 120BT0571

2 Satabdi Beuria 120BTO569
3 Ashna Reyaz 120BT0032

DATE OF EXPERIMENT DATE OF SUBMISSION

DEPARTMENT OF BIOTECHNOLOGY AND MEDICAL ENGINEERING

NATIONAL INSTITUTE OF TECHNOLOGY
ROURKELA-769008
ODISHA

MARKS
SIGNATURE REMARK
AWARDED
 DEPARTMENT OF BIOTECHNOLOGY & MEDICAL ENGINEERING
NATIONAL INSTITUTE OF TECHNOLOGY, ROURKELA
COURSE: BM3708 Analytical Techniques in Biotechnology Laboratory

ANALYTICAL TECHNIQUES IN
BIOTECHNOLOGY LAB (BM3708)
EXPERIMENT NO: 3

TITLE: UV-Vis spectrophotometry

DEGREE: B.Tech

SUBMITTED BY

GROUP- 1

SUBGROUP-5

SI NO. NAME REGISTRATION NO.

1 Karthik Peddi 120BT0571
2 Satabdi Beuria 120BT0569
3 Ashna Reyaz 120BT0032

DATE OF EXPERIMENT DATE OF SUBMISSION

DEPARTMENT OF BIOTECHNOLOGY AND MEDICAL ENGINEERING

 DEPARTMENT OF BIOTECHNOLOGY & MEDICAL ENGINEERING
NATIONAL INSTITUTE OF TECHNOLOGY, ROURKELA
COURSE: BM3708 Analytical Techniques in Biotechnology Laboratory

NATIONAL INSTITUTE OF TECHNOLOGY

ROURKELA-769008
ODISHA

EXPERIMENT-3
AIM: To study UV-Vis Spectrophotometry and determine the concentration
of an unknown sample using a standard curve.

THEORY:
UV-Vis spectrophotometry:
The ultraviolet-visible spectrophotometer is a type of ultraviolet
spectrophotometer. The UV-vis spectrophotometer is an analytical instrument
based on the principle of ultraviolet-visible spectrophotometry, which uses
substance molecules to analyze the absorption of radiation in the ultraviolet-
visible spectrum.
The Principle of UV-Visible Spectroscopy is based on the absorption of
ultraviolet light or visible light by chemical compounds, which results in the
production of distinct spectra. Spectroscopy is based on the interaction
between light and matter. When the matter absorbs the light, it undergoes
excitation and de-excitation, resulting in the production of a spectrum.
When matter absorbs ultraviolet radiation, the electrons present in it undergo
excitation. This causes them to jump from a ground state (an energy state with
a relatively small amount of energy associated with it) to an excited state (an
energy state with a relatively large amount of energy associated with it). It is
important to note that the difference in the energies of the ground state and
the excited state of the electron is always equal to the amount of ultraviolet
radiation or visible radiation absorbed by it.

Beer-Lambert Law
The statement of the Beer-Lambert law can be written as follows: When
a beam of monochromatic light is made incident on a solution that
contains a substance that absorbs the monochromatic light, the rate at
which the intensity of the beam decreases along the thickness of the
 DEPARTMENT OF BIOTECHNOLOGY & MEDICAL ENGINEERING
NATIONAL INSTITUTE OF TECHNOLOGY, ROURKELA
COURSE: BM3708 Analytical Techniques in Biotechnology Laboratory

solution is directly proportional to the concentration of the absorbing

substance in the solution and is also directly proportional to the intensity
of the incident monochromatic radiation.
As per the Beer-Lambert law, the greater the number of absorbing
molecules (that have the ability to absorb light of a specific wavelength),
the greater the extent of absorption of the radiation.
The instrumentation of a UV-vis spectrophotometer components:
1) Light source: A light source that emits light in the ultraviolet (UV)
and visible regions of the electromagnetic spectrum is used. The
most common light sources are deuterium lamps, which emit light in
the UV region, and tungsten-halogen lamps, which emit light in the
visible region.
2) Monochromator: The monochromator is used to select a specific
wavelength of light from the light source. This is done by passing the
light through a series of diffraction gratings or prisms, which
separates the light into its individual wavelengths.
3) Sample holder: A small, clear container called cuvette is used that
holds the sample in the light path of the spectrophotometer.
Cuvettes are typically made of glass or quartz, and they come in
different sizes and shapes depending on the type of
spectrophotometer and the type of sample being analysed.
4) Detector: The detector is used to measure the intensity of the light
that emerges from the sample. The most common types of detectors
are photomultiplier tubes PMT) and silicon photodiodes (PD).
5) Data acquisition system: The data acquisition system is used to
collect and store the data from the detector.
 DEPARTMENT OF BIOTECHNOLOGY & MEDICAL ENGINEERING
NATIONAL INSTITUTE OF TECHNOLOGY, ROURKELA
COURSE: BM3708 Analytical Techniques in Biotechnology Laboratory

METHODOLOGY
1)The sample was prepared by dissolving 100 mg of Salicylic acid in a 100 ml
solution containing 1 ml of 2-propanol and 99 ml water, to get a stock solution
of 1 mg/ml.
2)From this stock solution, solutions with concentrations of
0.001,0.002,0.0025,0.005,0.01,0.02,0.025 and 0.03 mg/ml were prepared.
3)The instrument was then calibrated by measuring these solutions, and a
standard curve was prepared, which would be used to determine the
concentration of the unknown sample.
4) The sample was placed in a cuvette and placed in the light path of the
spectrophotometer. The absorbance of the sample was measured at the
wavelength that gave maximum absorption, which turned out to be 294 nm in
this case.The measurement was done using a single-beam spectrophotometer.
5) The data obtained from the measurement was analysed to determine the
concentration of the sample. This was done by comparing the absorbance of
the sample to the standard curve.
OBSERVATION:

CONCENTRATION (mg/ml) ABSORBANCE (AU)

0.001 0.0249
0.002 0.0509
0.0025 0.077
0.005 0.1347
0.01 0.2584
 DEPARTMENT OF BIOTECHNOLOGY & MEDICAL ENGINEERING
NATIONAL INSTITUTE OF TECHNOLOGY, ROURKELA
COURSE: BM3708 Analytical Techniques in Biotechnology Laboratory

0.02 0.5208
0.025 0.6639
0.03 0.7928

Unknown sample absorbance value

U1 0.4814
U2 0.3957
U3 0.3957
U4 0.1916
U5 0.3088

RESULT:

CALCULATION:
The concentration of the unknown sample can be determined using the
equation obtained for the standard curve, y = 26.379 x.
-> x indicates the concentration of the solution in mg/ml
 DEPARTMENT OF BIOTECHNOLOGY & MEDICAL ENGINEERING
NATIONAL INSTITUTE OF TECHNOLOGY, ROURKELA
COURSE: BM3708 Analytical Techniques in Biotechnology Laboratory

-> y indicates its absorbance.

The absorbance of the unknown sample U5 was measured to be 0.3088
(y=0.3088).
X = y/26.379 = 0.3088/26.379
= 0.0117 mg/ml
Therefore, the concentration of unknown sample is 0.0117 mg/ml.

CONCLUSION:
In this Experiment we studied about the UV-visible Spectroscopy and we get to
know what is the working Principle of uv-visible spectroscopy.And known the
Application of uv-visible Spectroscopy.And we found the Concentration of
Unknown Sample.