Objective
Introduction
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
A = - log (%T)
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
A∝C
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.
A∝b
A ∝ bC
Thus,
A = єbc,
where є = molar absorptivity
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
Reagents
Apparatus
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
Equipment
Methods
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
Concentration Absorbance
(ppm) [A]
Blank 0 0.000
Std 1 5 0.193
Std 2 15 0.403
Std 3 25 0.564
Std 4 35 0.911
Std 5 45 1.321
Unknown
1 10.778 0.285
Unknown
2 19.740 0.527
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
y = 0.027x – 0.006
y = 0.027x – 0.006
0.285 = 0.027x – 0.006
x = 10.778 ppm
y = 0.027x – 0.006
0.527 = 0.027x – 0.006
x = 19.740 ppm
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
Discussions
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
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
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.
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
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.
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
Conclusion
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
Appendix
Sample Calculations
• 5 ppm
M1 V 1 = M2 V 2
(100ppm) (V1) = (5ppm) (50mL)
V1 = 2.5 mL
• 15 ppm
M1 V 1 = M2 V 2
(100ppm) (V1) = (15ppm) (50mL)
V1 = 7.5 mL
• 25 ppm
M1 V 1 = M2 V 2
(100ppm) (V1) = (25ppm) (50mL)
V1 = 12.5 mL
• 35 ppm
M1 V 1 = M2 V 2
(100ppm) (V1) = (35ppm) (50mL)
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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
V1 = 17.5 mL
• 45 ppm
M1 V 1 = M2 V 2
(100ppm) (V1) = (45ppm) (50mL)
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
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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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy
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