SINGLE BEAM UV-VIS SPECTROPHOTOMETER (SPECTRONIC)

Title

: The visible spectra of soft drinks

Objective

: a) To determine the wavelength at maximum absorbance ( max) a soft drink

sample
b) To determine the unknown concentration of a soft drink from the calibration
curve

Instrument : ThermoSpectronic/Gynesys 20
Standard

: Potassium permanganate (KmnO4)

Sample

: Potassium permanganaye (KmnO4)

Introduction :
The word spectroscopy is used to refer to the broad area of science dealing with the
absorption, emission, or scattering of electromagnetic radiation by molecules, ions, atoms, or
nuclei. Spectroscopic techniques are some of the most widely used analytical methods in the
world today. These techniques are useful in determining both the identity of unknown substances
and their concentration in solution. Different regions of the electromagnetic spectrum such as
infrared, visible, ultraviolet, or, X-ray radiation can be used to interact with matter. The
Spectronic 20 instrument you will use can correctly be called a colorimeter, because it measures
the absorption of light in the visible spectrum that we perceive as color, and the technique used is
said to be colorimetric. The electrons and nuclei of atoms and molecules may exist in only
certain specific energy levels, that is, they area quantized. They can absorb only photons having
certain energies or wavelengths. The energies of light absorbed by a molecule can be related to
motions (energy modes) of the molecule.
When a beam of light of a given wavelength strikes the sample tube in a
spectrophotometer, part of it is absorbed by the sample, part of it is transmitted to the detector,
and part of it is lost through the reflection from the tubes surfaces, refraction (bending) by the
solution, or scattering from small particles suspended in the sample. When we allow the light of
the same given wavelength to pass through a blank -- identical to the sample in every way
except for the presence of the light absorbing species of interest -- all of these processes except
absorption by the sample occur. Transmittance, T, is the ratio of intensity of light passing through
a sample at a given wavelength divided by the intensity of light passing through a blank at the
same wavelength. That is:
T = Ix/Io

where Ix is the intensity of light passing through a sample and Io is the intensity of light passing
through a blank. Percent transmittance is merely 100 times the transmittance: % T = 100 x T.
Another parameter of interest in a spectophotometric experiment is absorbance, A.
Absorbance = A = - log T
Both transmittance and absorbance are unitless quantities. As it turns out, the absorbance of a
sample can be related rather neatly to some relatively simple parameters through an equation
known as the BeerLambert law or simply Beers Law:
A=al c
where A is still Absorbance, l is the path length of light through the sample cell (1 cm in a
standard Genisys 20 tube), and a is the Absorptivity. The absorptivity is the proportionality
constant in Beers Law, which varies from one light absorbing species to another and has a
wavelength dependence, which is also different from one light absorbing species to another.
Thus, the value of A can be changed by one of the following ways for any given light absorbing
species: (a) change the wavelength, (b) change the pathlength, or (c) change the concentration.
The pathlength is usually fixed at some convenient value through spectrophotometer and sample
cell design. Also, the absorptivity is usually specified only at max which is the wavelength at
which light is most strongly absorbed by the light absorbing species. It is worth noting that at for
a species of a given concentration in a cell of given path length the variation of A per unit change
in concentration is most dramatic because absorptivity is greatest at max. Therefore, at max
the concentration of a light absorbing species can be determined by comparing light absorption
of solution of unknown concentration to the absorption of samples of known concentration. This
is done by making a graph of A vs. concentration for the standards (sometimes referred to as a
Beers Law plot or a standard curve) and then finding what concentration corresponds to the
value of A observed for the unknown.

Methods :
A) Preparation of standard solution of soft drink (known concentration)
1. Soft drink was poured into beaker. It was then stirred to remove carbonation.
2. Stock solution (50%) 125ml (soft drink) was pipetted into 250ml volumetric flask. The
volumetric flask was covered and shaked homogenously.
3. 40%, 30%, 20% and 10% standard solution was prepared from stock solution (40ml,
30ml, 20ml and 10ml) using M1V1=M2V2.
B) Operation of the Spectronic 20 and determination of max
1. Spectronic 20 was turned on. The instrument was waited to wake up for 15 minutes
(warm up).
2. Wavelength was set at 600nm.
3. 0% transmittance (%T) was adjusted. Nothing should be in the sample compartment.
4. Cuvette was cleaned and rinsed. The cuvette was filled until 2/3 full of blank (deionized
water).
5. 0 absorbance and 100%T were adjusted.
6. Cuvette was rinsed with standard. The cuvette was filled until 2/3 full of standard. The
absorbance was read and recorded.
7. The cuvette was removed and top equiment was closed. Wavelength was changed to
20nm lower and 100%T was reset if it changed.
8. The step was repeated until 360nm.
9. Absorbance spectrum was plotted and th max was determined.
C) Preparation of unknown soft drink sample
1. Soft drink was poured into beaker. It was then stirred to remove carbonation.
2. The soft drink was poured into 50ml volumetric flask (dilute) without measure the
volume.
3. The unknown sample was put into cuvette until 2.3 full.
D) Quantitative analysis of the soft drink solution
1. Wavelength maximum (max) was set.
2. 0 ab 100% T was set.
3. Absorbance of each 5 standard solution and unknown solution was measured and
recorded.

Results :

The wavelength and absorbance of soft drink

Wavelength (nm)
600
580
560
540
520
500
480
460
440
420
400
380
360

Absorbance
0.054
0.072
0.161
0.121
0.290
0.207
0.080
0.058
0.030
0.029
0.037
0.050
0.089
max = 520nm

Table of samples concentration (ppm) and absorbance

Solutions
1
2
3
4
Unknown sample

Calculation :

Concentration (ppm)
5
10
15
20
1.9

Absorbance (nm)
0.073
0.158
0.231
0.295
0.145

Dilution of standard solution :

50% stock solution :
M1V1 = M2V2
100(V1) = (50)(250)
V1 = 125ml of stock solution
10%

M1V1 = M2V2
(50)V1 = (10)(50)
V1 = 10ml of stock solution

20%

M1V1 = M2V2
(50)V1 = (20)(50)
V1 = 20ml of stock solution

30%

M1V1 = M2V2
(50)V1 = (30)(50)
V1 = 30ml of stock solution

40%

M1V1 = M2V2
(50)V1 = (40)(50)
V1 = 40ml of stock solution

Concenteration of unknown sample :

y = mx + c
y = 0.0739x + 0.0045
0.145 = 0.0739x + 0.0045
X = 1.9 ppm

Discussion :

For this experiment, we use soft drinks as a sample. The objective the experiment is to determine
the wavelength at the maximum absorbance a soft drink sample and determine the unknown
concentration of a soft drink from the calibration curve.
Spectroscopy is the study of interaction between electromagnetic radiation and atoms,
molecules and other chemical apecies. Spectroscopic methods are based on te absorption or
emission of radiation in ultraviolet (UV), visible (Vis), infrared (IR) and radio (nuclear magnetic
resonance,NMR). Detection of food constituents can be achieved by measuring the amount of
radiation. The type of electromagnetic radiation used in this experiment is visible light. Visible
light is the only part of the electromagnetic spectrum that humans can see with an unaided eye.
This part of the spectrum includes a range of different colors that all represent a particular
wavelength. Rainbows are formed in this way; light passes through matter in which it is absorbed
or reflected based on its wavelength. Thus, some colors are reflected more than other, leading to
the creation of a rainbow. Transmittance, T, is the ratio of the light transmitted through the
sample and the light entering the sample while absorbance, A, is the amount of light absorbed
from the initial incoming light. Absorbance is the base 10 logarithm of the reciprocal of
transmittance. Beer-Lambert Law stated that the amount of light absorbed by a solution is
proportional to the concentration of the solution and to the length of the solution.

Log lo / lt = abc (or lc)

Where:
lo : intensity of incident light
lt : intensity of transmitted light
a : molar absorptivity of solute measured (1 mol-1 cm-1)
b : length of light path or length of solution (cm)
c : concentration of solute in solution

The path length is usually fixed at some convenient value through spectrophotometer and sample
cell design. Also, the absorptivity is usually specified only at max which is the wavelength at
which light is most strongly absorbed by the light absorbing species. Based on Beers Law, the
absorbance will increase linearly with an increase in concentration and the transmittance will
decrease exponentially with an increase in concentration. It is important to first obtain the
absorption spectrum of the soft drink before making a calibration curve to determine the
wavelength of maximum absorption. The absorbance spectrum shows how the absorbance of
light depends upon the wavelength of the light. The spectrum itself is a plot of absorbance vs
wavelength and is characterized by the wavelength (max) at which the absorbance is the
greatest. A blank is used in order to cancel out or zero the absorbance of all the other components
in the sample except the component whose absorbance is to be measured.

Conclusion :
The concentration of unknown soft drink sample is 1.9 ppm.

References :
1. John M. Chalmers; Peter Griffiths, eds. (2006). Handbook of Vibrational Spectroscopy.
New York: Wiley.
2. Jerry Workman; Art Springsteen, eds. (1998). Applied Spectroscopy. Boston: Academic
Press.
3. Symmetry and Spectroscopy: An Introduction to Vibrational and Electronic Spectroscopy
(Paperback) by Daniel C. Harris, Michael D. Bertolucci.
4. James D. Ingle, Jr. and Stanley R. Crouch, Spectrochemical Analysis, Prentice Hall, 1988