FLOW CHART

To prepare a dilution,
measure exact volume
Prepare serial dilutions Calculate volume
of V1 and pour into 50
(5 ppm, 15 ppm, 25 needed, V1 from
ml volumetric flask.
ppm, 35 ppm and 45 the 100 ppm food
Then add distilled
ppm) in 50 ml dye stock using
water until the
volumetric flask. dilution formula. meniscus Then, shake
volumetric flask
properly.

Fill a cuvette with 45

ppm dilution and
Instructor will brief
another cuvette with
on operating
blank solution, insert
procedure of Perkin- Repeat the process
them in sample
Elmer UV/Vis for all dilutions.
compartment. Wipe
Spectrophotometer
clean sides of cuvettes
Lambda EZ210.
but avoid touching
clear surface. Do
wavelengths scan and
obtain max. Record
data.

For photometric scan,

fill cuvette as before
but use the serial Determine the Record all data
dilution prepared and concentrations of completely and
scan one by one. Unknown 1 and clean workstation
Record absorbance Unknown 2. properly.
reading and look at
standard calibration
graph produced.
Analysis of Data:

Record different
Use the spectrum obtained concentration of standards
from wavelength scan to absorbance and construct
identify max for Carmoisine standard calibration curve.
and then record. (Concentration vs
Absorbance)

Measure Unknown 1 and

Unknown 2 concentration
peak in the soda
samplechromatograph, and
use standard calibration curve
to determine concentration of
Unknown 1 and 2.
 RESULTS

Part A: Wavelength Scan

(i) Instrument Parameters
Starting Wavelength = _______700.0__________ nm
Ending Wavelength = ________400.0_________ nm
Path length = ________10.0_________ mm

(ii) Result for Wavelength Scan

Sample used = _____________Carmoisine stock____
Concentration of the sample = _______100__________ ppm
 max obtained = _____540____________ nm

Part B: Photometric Scan

(i) Instrument Parameters
Wavelength,  max = _____537.0____________ nm
Path length = _______10.0___________ mm

(i) Results for Photometric Scan (Creating a Standard Calibration Curve)

Table 1: Data
No. Sample Carmoisine Concentration Absorbance (nm), y-axis
(ppm), x-axis

1 Std 1 5.000 0.091

2 Std 2 15.000 0.242

3 Std 3 25.000 0.416

4 Std 4 35.000 0.592

5 Std 5 45.000 0.720

6 Unknown 1 20.614 0.338

7 Unknown 2 42.180 0.692

 DISCUSSION

In the analysis of food colour experiment, there are 2 objective that need to be focus on.
The first objective was to determine λmax of Colourant (wavelength scan) and the second objective
was to prepare a serial dilution and generate a standard calibration graph for sample quantification.
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. The 100ppm stock Carmoisine
solution was been diluted to 5 different concentration which are 5pm, 15ppm, 25ppm, 35ppm and
45ppm. When analyzing by using UV-VIS Spectrophotometer, the blank solution used was
distilled water. For sample solution, the technician prepared the student with two samples for
analyzing it using UV-VIS Spectrophotometer.

The experiment was continuing by putting the sample in a cuvette. A cuvette is a small
tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz
(for UV light) and designed to hold samples for spectroscopic experiments. Disposable plastic
cuvettes are often used in fast spectroscopic assays, where speed is more important than high
accuracy. Some cuvettes will be clear only on opposite sides, so that they pass a single beam of
light through that pair of sides; often the unclear sides have ridges or are rough to allow easy
handling. Cuvettes to be used in fluorescence spectroscopy must be clear on all four sides because
fluorescence is measured at a right-angle to the beam path to limit contributions from beam itself.
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.

The sample was then been tested using the instruments. Two types of analysis were done,
which are, wavelength scanning and photometric scanning. λmax was obtained by scanned the
highest concentration of the dilution which are 45ppm. For the photometric scan, the different
dilution of sample was been scan to produce 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. The data for absorbance and
concentration can be found in Table 1. From the result obtained and the graph that has been plotted,
the Standard Calibration Graph line is linear related to the Beer’s Law. So, that means, from this
experiment we know that Beer’s Law theory is true that the relationship between the absorbance
of the solution and the concentration at the absorbing species have been proved.
 CONCLUSION

UV spectrophotometer is a device used to study the interaction between radiation and

matter in regards to the wavelength of photons. 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 o the spectrum. Ultra-Visible Spectrophotometer is used in this experiment
to determine the maximum wavelength of Carmoisine solution. The 100ppm stock Carmoisine
solution was been diluted to 5 different concentration which are 5pm, 15ppm, 25ppm, 35ppm and
45ppm. The sample was then been tested using the instruments. Two types of analysis were done,
which are, wavelength scanning and photometric scanning. From the result obtained, a standard
calibration curve was plotted. The graph that was plotted is linear so that proves the Beer’s Law
Theory is true that the relationship between the absorbance of the solution and the concentration
at the absorbing species have been proved.
 RECOMMENDATION

Some recommendations can be done in this experiment to improve its accuracy in the
future. For example, when doing the dilution process, we must make sure to measure the exact
volume needed from the 100 ppm food dye stock and make sure to fill up the volumetric flask with
distilled water until the meniscus then shake it properly. Next, when wiping the sides of the cuvette,
make sure not to touch the clear surface because it will affect the final results. Finally, make sure
to operate the Perkin-Elmer UV/Vis Spectrophotometer Lambda EZ210 with the right procedure
to avoid errors on the results and graph.
 TUTORIALS

1. State the Beer’s Lambert Law.

A= bc
Where A is absorbance and it does not have units, is the molar absorptivity with units
of L mol-1 cm-1. b is the path length of the cuvette in which contain the sample. The unit
is in centimeters. c is the concentration of the compound in solution, expressed in M or
mol L-1 .

2. What is the volume needed to prepare a 50 ppm of carmoisine from a 100 ppm of
carmoisine in 100 ml volumetric flask?

M1 V1 = M2 V2
(100ppm) (V1) = (50ppm) (100mL)
V1 = 50 mL

3. Why we need to wipe the sides of the cuvette clear surface?

Cuvettes to be used in fluorescence spectroscopy must be clear on all four sides because
fluorescence is measured at a right-angle to the beam path to limit contributions from
beam itself. 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.

4. Describe the function of wavelength scanning and photometric scanning.

The function of wavelength scanning is to detect things and to understand them in a

better way. It is like an x-ray. Most of the time scanning refer to health and technologist.
They can also have something to do with science or technology. It is also done to
determine at what wavelength the carmoisine able to absorb in the range of 200 nm to
700 nm which we cannot seen by our vision.

The use of photometric scan is to determine the concentration of an unknown sample,

after getting a standard curve from a series of known concentration. In this experiment,
there is two unknown sample that is use.
 REFERENCES

1. Food Analysis, Third Edition, Kluwer Acedemic/Plenum Publishers, S. Suzanne

Nielsen, 2003, New York, 2003

2. Darrel D. Ebbing, Steven D. Gammon, General Chemistry Ninth Edition, Houghton

Mifflin Company (2009).

3. http://elchem.kaist.ac.kr/vt/chem-ed/spec/uv-vis/uv-vis.htm , Uv-Vis Spectroscopy

4. https://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy ,
Ultraviolet-visible Spectroscopy

5. Drisko,R.L Chemistry 108 LabTextbook.N.P. Pauline M. Hamilton,2002,Print Food

Dye Chromotoagraphy “Flinn Scientific INC,8 August,2002,Web. 15 Sept. 2014