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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Objective
1. To determine λmax for Carmoisine sample (wavelength scan)
2. The prepare a serial dilution and generate a standard calibration graph for
sample quantitation (photometric scan)
Introduction
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-VIS)
involves the spectroscopy of photons and spectrophotometry. It uses light in visible and
adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this region of energy
space molecules undergo electronic transitions. Electromagnetic radiation in th UV-VIS
portion of the spectrum ranges in wavelength from approximately 200 to 700 nm. The
UV range is colorless to the human eye, while different wavelengths in the visible range
each have the characteristic color, ranging from violet at the short wavelength end of
the spectrum to red at the long wavelength end o the spectrum.
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Figure 1 : Electromagnetic Spectrum
Figure 2 : Visible Spectrum
The instrument used in ultraviolet-visible spectroscopy is called Ultraviolet-Visible
Spectrophotometer. It measures the intensity of light passing through a sample (I), and
it compares it to the intensity of light before it passes through the sample (Io). The ratio
I/Io is called the transmittance, and is usually expressed as a percentage (%T). The
absorbance, A, is based on the transmittance :
A = - log (%T)
Figure 3 : One kind of Ultraviolet Visible Spectroscopy
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Like Colorimeter and Atomic Absorption Spectroscopy (AAS), UV-VIS also applies Beer-
Lambert Law, which is the combination of Beer’s Law and Lambert’s Law. Beer’s Law is
defined as the absorbance (A) is directly proportional to concentration of solution (C)
when a beam of monochromatic light is passed through a solution of constant length.
Lambert’s Law is defined as the absorbance (A) is directly proportional to thickness of
solution (b) when beam of monochromatic light is passed through a solution of constant
concentration.
Combining Beer’s and Lambert’s expression, we have :
Thus,
Figure 4 : UV-VIS Schematic Diagram
A C
A b
A bC
A = єbc,
where є = molar
absorptivity
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Reagents
100ppm Carmoisine stock (100ml)
Unknown concentration of Carmoisine (2 samples)
Distilled water
Apparatus
Sample cuvettes, path length 1 cm
Volumetric flasks 50mL (five)
Pipette 5 ml, 10 ml, 25 ml (one each)
Rubber bulb (three)
Beaker 100 ml (one)
Graduated cylinder 50 ml (one)
Dropper (one)
Labeling sticker
Tissue paper
Equipment
Perkin Elmer UV-Vis Spectrophotmeter Lambda EZ210
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Methods
1. Serial dilutions (5ppm, 15ppm, 25ppm, 35 ppm, 45ppm) from the 100ppm
carmoisine stock were prepared.
2. The volume needed, V1 from the 100ppm carmoisine stock was calculated for all
dilutions.
3. In order to prepare a dilution, an exact volume of V1 was drew from the
carmoisine stock and was poured into a 50ml volumetric flask. Distilled water
then was added up to the mark level of the volumetric flask. The volumetric flask
then was shook properly.
4. The procedure previous was repeated for all dilutions. The formula used is :
M1 V1 = M2 V2 to find the V1
Where M1 = concentration of carmoisine stock
V1 = volume of carmoisine stock to be drawn
M2 = concentration of carmoisine (diluted)
V2 = volume of carmoisine (diluted)
5. After preparing the serial dilutions, the technician briefed on the standard
operating procedure of Perkin Elmer UV-VIS Spectrophotometer Lambda EZ210.
6. A cuvette was filled with 45pm dilution and another cuvette was filled with blank
solution, then the cuvettes were inserted in the sample compartment. The clean
sides of the cuvettes were wiped clean and not touched. The wavelength scan
was done and the λmax was obtained. The data was recorded.
7. For the photometric scan,the cuvette was filled as step 6 but the serial dilution
prepared was used and scanned one by one. The absorbance readings were
recorded ant the standard calibration graph produced was analyzed.
8. The concentrations of two unknown solutions were determined.
9. Work station was cleaned properly before leaving the laboratory.
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Results
Table 1 : Dilution Factors and Absorbance of Carmoisine
Concentration (ppm) Absorbance [A]Blank 0 0.000Std 1 5 0.193Std 2 15 0.403Std 3 25 0.564Std 4 35 0.911Std 5 45 1.321
Unknown 1 10.778 0.285Unknown 2 19.740 0.527
0 5 10 15 20 25 30 35 40 45 500.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
f(x) = 0.027467397260274 x − 0.00690410958904109R² = 0.976427141469739
Absorbance [A] vs Concentration (ppm)
Concentration (ppm)
Abso
rban
ce [A
]
Figure 5 : Absorbance vs Concentration (Standard Calibration Graph for
Carmoisine)
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
The standard calibration curve is obtained with the standard deviation of 0.976
and the linear regression equation is :
y = 0.027x – 0.006
Since the value of absorbance, [A] of the unknown solution is represented as y in
the equation, the concentration of the unknown solutions can be calculated :
Unknown 1 (Absorbance, [A] = 0.285)
y = 0.027x – 0.006
0.285 = 0.027x – 0.006
x = 10.778 ppm
Unknown 2 (Absorbance, [A] = 0.527)
y = 0.027x – 0.006
0.527 = 0.027x – 0.006
x = 19.740 ppm
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Discussions
Ultra-Visible Spectrophotometer is used in this experiment to determine the maximum
wavelength of Carmoisine solution. Carmoisine is one of permitted colors that can be
used in food. It is red in color, which is natural that usually used as colorant in jellies.
In this experiment, the stock solution of 100ppm Carmoisine is diluted into 5 serial
dilutions of 5ppm, 15ppm, 25ppm, 35 ppm, 45ppm. For sample solutions, we randomly
mixed 2 serial dilutions into one and did the same way for the second sample solution.
When analyzing by using UV-VIS Spectrophotometer, the blank solution used was
distilled water.
The cuvettes used in the instrument are the most important part to be taken care of.
The cuvette has 2 different surfaces, where the rough ones can be touched by bare
fingers and the other ones, which are the smooth ones shouldn’t be touched by fingers.
This is because the smooth sides of the cuvette are where the light will go through the
sample from the source. If the smooth sides of cuvette were stick with fingerprints, the
light might be diffused to another way. That was why wiping the smooth surfaces of the
cuvette is very important.
The instrument was run by the technician. There were two types of scanning done –
wavelength scanning and photometric scanning. To obtain the λmax for Carmoisine
sample, the 45ppm dilution which is the highest concentration solution was scanned
and the wavelength scan was done. For photometric scan, each dilution were scanned
to produce the standard calibration graph.
The data of results consist of the concentration values of the five standards with their
respective absorbance with a standard calibration graph and the standard deviation.
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
The concentration of the unknown samples also were automatically computed and
printed on the data of results. Although the concentration of unknown solutions has
been obtained by the instrument, manual calculations still been done for comparisons.
After obtaining the data of results, the linear calibration graph were re-plotted manually
to obtain the equation of linear regression using Microsoft Office Excel software. The
equation obtained with standard deviation of 0.976 is :
y = 0.027x – 0.006
Since the value of absorbance, [A] of each of the unknown solutions are represented as
y in the equation, the concentration of the unknown solutions can be calculated where:
Unknown 1 : 10.778 ppm
Unknown 2 : 19.740 ppm
The manually calculated values of results are slightly different than the results obtained
automatically by the instrument due to the calibration that may have been done on the
instrument.
The maximum wavelength in the experiment was obtained 510nm.
There were no problems occurred while running the experiment.
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Conclusion
The experiment was successfully done and the objectives of the experiment are
achieved. The concentrations of two unknown solutions had been calculated to be
10.778 ppm and 19.740 respectively. The maximum wavelength, λmax for Carmoisine
sample is 510nm.
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
Appendix
Sample Calculations
Preparation of Serial Dilutions
5 ppm
M1 V1 = M2 V2
(100ppm) (V1) = (5ppm) (50mL)
V1 = 2.5 mL
15 ppm
M1 V1 = M2 V2
(100ppm) (V1) = (15ppm) (50mL)
V1 = 7.5 mL
25 ppm
M1 V1 = M2 V2
(100ppm) (V1) = (25ppm) (50mL)
V1 = 12.5 mL
35 ppm
M1 V1 = M2 V2
(100ppm) (V1) = (35ppm) (50mL)
V1 = 17.5 mL
45 ppm
M1 V1 = M2 V2
(100ppm) (V1) = (45ppm) (50mL)
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CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy
V1 = 22.5 mL
Data of Results
(attached in the end of the report)
References
Books
Food Analysis, Third Edition, Kluwer Acedemic/Plenum Publishers, , S. Suzanne
Nielsen, 2003, New York, 2003
Lecture Notes
The Influences of Color in the Acceptance of Jellies, Nadiah bt Mohd Kahar,
UniKL MICET, 2007
Websites
http://elchem.kaist.ac.kr/vt/chem-ed/spec/uv-vis/uv-vis.htm
http://en.wikipedia.org/wiki/Ultraviolet-visible_spectroscopy
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
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