CFB 40103 PRACTICAL

MALAYSIAN INSTITUTE OF CHEMICAL AND BIOENGINEERING

TECHNOLOGY.

ADVANCED FOOD ANALYSIS (CFB 40103)

FOOD ANALYSIS 2 (CFB 40403)
UNIKL

LAB MANUAL

DOC. NO. :

MICET
JAN 07
(REV. NO. 1)

PRACTICAL 2: UV-VIS SPECTROSCOPY

OBJECTIVE
1.
2.
3.

To determine max for carmoisine sample (wavelength scan)

To determine max for carbonated drink sample (wavelength scan)
To prepare a serial dilution and generate a standard calibration graph for sample quantitation
(photometric scan)

INTRODUCTION
Absorption of light by solution is one of the oldest and still one of the more useful instrumental methods. The
wavelength of light that a compound will absorb is characteristic of its chemical structure. Specific regions of the
electromagnetic spectrum are absorbed by exciting specific types of molecular and atomic motion to higher
energy levels. Absorption of microwave radiation is generally due to excitation of molecular rotational motion.
Infrared absorption is associated with vibrational motions of molecules. Absorption of visible and ultraviolet
(UV) radiation is associated with excitation of electrons, in both atoms and molecules, to higher energy states. All
molecules will undergo electronic excitation following absorption of light, but for most molecules very high
energy radiation (in the vacuum ultraviolet, <200 nm) is required. For molecules containing conjugated electron
systems however, light in the UV-visible region is adequate (e.g., benzene absorbs in the 260 nm region). As the
degree of conjugation increases, the spectrum shifts to lower energy.
Electromagnetic radiation in the 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 of the spectrum. Because absorption spectra are characteristic of molecular structure, they can be
used to qualitatively identify atomic and molecular species.

The amount of light, I, transmitted through a solution of an absorbing chemical in a transparent solvent can be
related to its concentration by Beers Law:

-Log I/Io = A=bc

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CFB 40103 PRACTICAL

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
Methods
1. Prepare serial dilutions (5ppm, 15ppm, 25ppm, 35 ppm, 45ppm) from the 100ppm carmoisine stock.
2. Calculate volume needed, V1 from the 100ppm carmoisine stock for all dilutions.
3. In order to prepare a dilution, an exact volume of V1 was drawn up from the carmoisine stock and was
poured into a 50ml volumetric flask. Add distilled water up to the mark level of the volumetric flask.
4.

Shake the volumetric flask properly.

The procedure previous was repeated for all dilutions. The formula used is :
M1V1= M2V2
Where

to find the V1

M1= concentration of carmoisine stock

V1= volume of carmoisine stock to be drawn
M2= concentration of carmoisine (diluted)
V2= volume of carmoisine (diluted)

5.

Your instructor will brief on the standard operating procedure of Perkin Elmer UV-VIS

6.

Spectrophotometer Lambda EZ210.

Fill a cuvette with 45ppm dilution and another cuvette with blank solution. Insert both cuvettes in the
sample compartment and blank compartment. Wipe clean the clean sides of the cuvettes and not

7.

touched. Conduct wavelength scan was to obtain the max. Record data obtained from this scan.
For the photometric scan, the cuvette was filled as step 6 but the serial dilution prepared was used and

8.
9.

scanned one by one. Record all absorbance readings and prepare the standard calibration graph.
Determine the concentrations of two unknown solutions.
Clean your work station before leaving the laboratory.

Result
Plot a calibration curve for the standard solutions to demonstrate that the absorbance follows Beer's Law. Perform
a linear regression to determine the slope of the line and its standard deviation. Determine the amount of
carmoisine in the unknown sample in mg/L based on the absorbance of both sample solutions.
DATA
Date:
Product information:

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CFB 40103 PRACTICAL

Absorbance of standards and samples:

Absorbance
Standards
(ppm carmoisine/ml)
0 (blank)
5
15
25
35
45

Msmt 1

Msmt 2

Average

Samples
Rep 1
Rep 2
Discussion & Conclusion
Calculation of total carmoisine in sample:
1.

Plot the standard curve and determine the content of carmoisine (g carmoisine/ml, ppm and ppb) in the
dissolved ash solution.

2.

Calculate the carmoisine (g carmoisine /g, ppm and ppb) in the sample.

QUESTION
1. What would be the advantages and disadvantages of the instrumental method versus the other method you
identified?
2. What is the maximum absorbance for carmoisine?
3. For a particular assay, your plot of absorbance vs. con-centration is not linear. Explain the possible reasons
for this.
4. A particular food coloring has a molar absorptivity of 3.8103cm1M1 at 510 nm.
(a) What will be the absorbance of a 2104M solution in a 1-cm cuvette at 510 nm?
(b) What will be the percent transmittance of the solution in (a)

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