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CFB 40103 – Advance Food Analysis

Practical 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 Analysis
Practical 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)

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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy

Figure 3 : One kind of Ultraviolet Visible 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.

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

Combining Beer’s and Lambert’s expression, we have :

A ∝ bC
Thus,

A = єbc,
where є = molar absorptivity

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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy

Figure 4 : UV-VIS Schematic Diagram

 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

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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy

 Equipment

Perkin Elmer UV-Vis Spectrophotmeter Lambda EZ210

 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 V 1 = M2 V 2 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)

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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy

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.
 Results

Table 1 : Dilution Factors and Absorbance of Carmoisine

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

Figure 5 : Absorbance vs Concentration (Standard Calibration


Graph for Carmoisine)

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 Analysis
Practical 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

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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.

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. 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.

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CFB 40103 – Advance Food Analysis
Practical 4 : UV-VIS Spectroscopy

The maximum wavelength in the experiment was obtained 510nm.

There were no problems occurred while running the experiment.

 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 Analysis
Practical 4 : UV-VIS Spectroscopy

 Appendix

Sample Calculations

Preparation of Serial Dilutions

• 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
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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

• 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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CFB 40103 – Advance Food Analysis
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