EXPERIMENT 2
ANALYSIS OF FOOD COLOUR
1.0 Objective
The objective of this experiments was to determine max of Carmoisine ( wavelength
scan )
To prepare a serial dilution and generate a standard calibration graph for sample
quantitation ( photometric scan )
2.0 Summary
3.0 Introduction
The purpose of this experiment was to analyze the food colouring by using an
ultraviolet/visible spectrometer. UV spectrometer is a device used to study the interaction
between radiation and matter in regards to the wavelength of photons. Specifically, it measure
visible light and the close-to-visible range of ultraviolet and infrared spectrum ranges. The
UV spectrometer allows a user to identify electronic transitions within the various regions of
the electromagnetic spectrum. Food colouring are mainly used in the food processing
industry today as colour gives the food product certain flavours as people associate colours
with certain flavours.
A spectrophotometer can be either single beam or double beam. In this experiment a
double beam spectrometer was used .In a double-beam instrument, the light is split into two
beams before it reaches the sample.
The Beer-Lambert law states that the absorbance of a solution is directly proportional to the
concentration of the absorbing species in the solution and the path length. Thus, for a fixed
path length, UV/Vis spectroscopy can be used to determine the concentration of the absorber
in a solution. Absorption spectroscopy in the UVVIS region is based on the Lambert-Beers
law, expressed by the following equations :
A=bc
Where;
A = absorbance (no unit)
= molar absorptivity coefficient (M-1 cm-1)
= pathlength (cm)
= concentration (M or mol/L)
4.0 Materials and Methodology
4.1 Materials:
Chemicals
a) 100 ppm colorant stock (100ml)
b) Unknown ( two )
c) Distilled water
Apparatus
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
Perkin Elmer UV/Vis Spectrometer Lambda EZ210
Sample Cuvettes
Path length 1 cm
Volumetric flask 50 ml (five)
Pipette 5 m, 10 ml, 25 ml
Rubber bulb (three)
Beaker 100 ml
Graduated cylinder 50 ml
Dropper
Labelling sticker
Tissue paper
5.2 Discussion:
The objective of the experiment was to determine max of Carmoisine ( wavelength
scan ) and to prepare a serial dilution and generate a standard calibration gaph for sample
quantitation (photometric scan ). In this experiment, the stock solution of 100ppm Colourant
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 shouldnt 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 max 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 replotted manually to obtain the equation of linear regression using Microsoft Office Excel
software. The equation obtained with standard deviation of ???? is :
y =??x ??
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 ?????nm. There were
no problems occurred while running the experiment.
9.0 Appendices
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 m
25 ppm
M1 V1= M2 V2
(100ppm) (V1) = (25ppm) (50mL)
V1= 12.5 m
35 ppm
M1 V1= M2 V2
(100ppm) (V1) = (35ppm) (50mL)
V1= 17.5 m
45 ppm
M1 V1= M2 V2
(100ppm) (V1) = (45ppm) (50mL)
V1= 22.5 m
8.0 References
Food Analysis, Third Edition, Kluwer Acedemic/Plenum Publishers, , S.Suzanne
Nielsen, 2003, New York, 2003
Lecture Notes
Ultraviolet-Visible Spectroscopy . Available from
https://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy
6.0 Conclusion:
Based on the experiment done, the experiment was successfully done and the
objectives of the experiment are achieved to determine max of Carmoisine ( wavelength
scan ) and to prepare a serial dilution and generate a standard calibration graph for sample
quantitation (photometric scan ). The concentrations of two unknown solutions had been
calculated to be ???ppm and ????ppm respectively. The maximum wavelength, max for
Carmoisine sample is ???nm.
4.2 Methodology
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 max 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.
5.0 Result and discussion
5.1 Results
7.0 Tutuorial
7.1 Pre Laboratory Questions
