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INTRODUCTION

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


matter in regards to the wavelength of photons. Specifically, it measures visible light and the
close-to-visible range ultraviolet and infrared spectrum ranges. The UV spectrosphotometer
allows a user to identify electronic transitions within the various regions of the electromagnetic
spectrum. 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.
Spectrophotometry is a very important and useful tool, which involves the interaction of
matter with electromagnetic radiation (EM). The spectrophotometer that you will use in this
experiment measures the visible portion of the EM spectrum, from 400-800 nm (1 nm = 10-9 m).
The spectrophotometer will be used to find the absorption of a food dye at several different
concentrations and then used to determine the unknown concentration of the same food dye.

Figure 1: Visible Spectrum


Traditionally, a spectrophotometer cannot detect fluorescence. This requires an additional
component known as a bi-spectral fluorescent mechanism. Without this ability, it is difficult to
properly manage color imagery, specifically if the color contains some sort of fluorescence. One
advantage to this device is the fact that it can identify the exact levels of compounds within a
particular spectrum sample. For example, if it analyzes a photograph, it should be able to identify

the different color components of each section of the image. Each color and the saturation of the
color is identifiable.
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 of:
A = - log (%T)

METHODS

1) From the 100 ppm Carmoisine stock, a serial dilution


which is 5 ppm, 15 ppm, 25 ppm, 35 ppm, and 45 ppm was
prepared.

2) 45 ppm dilution was


filled into a

cuvette

and

another

cuvette

with

blank

cuvettes

was

inserted in the sample

solution,
sides

of

the

compartment.

The

cuvettes were

wiped clean and the

clean surface

cannot

wavelength

scan is done and the

touch.

the
The

max is obtained.

3) The cuvette was filled as step no.2 but used


the serial dilution prepared and scans one by
one for the photometric scan. The absorbance
reading

is

recorded

and

calibration graph is produced.

the

standard

RESULT
A) The Preparation of Serial Dilutions

STOCK

DILUTED

Concentration
of Carmoisine
stock (ppm)

Volume of Carmoisine stock (ml)

Concentration
of Carmoisine
stock (ppm)

Volume of
Carmoisine stock
(ml)

M1

V1

M2

V2

100

(100) V1 = (5)(50)
V1 = (5)(50)
100
= 2.5 ml

50

100

(100) V1 = (15)(50)
V1 = (15)(50)
100
= 7.5 ml

15

50

100

(100) V1 = (25)(50)
V1 = (25)(50)
100
= 12.5 ml

25

50

100

(100) V1 = (35)(50)
V1 = (35)(50)
100
= 17.5 ml

35

50

100

(100)V1 = (45)(50)
V1 = (45)(50)
100
= 22.5 ml

45

50

B. Wavelength Scan
(1)

Instrument Parameters
Starting Wavelength

= 700.0 nm

Ending Wavelength

= 400.0 nm

Path length

10.0 mm

Standard no.

Concentration (ppm)

Absorbance

Blank

0.000

Std 1

0.093

Std 2

15

0.281

Std 3

25

0.469

Std 4

35

0.650

Std 5

45

0.830

Unknown 1

14.710

0.274

Unknown 2

29.133

0.540

(2)

Result for Wavelength Scan


Sample used

= Carmoisine stock

Concentration of the sample = 45 ppm


max obtained

= 515.0 nm

C. Photometric Scan
(1)

Instrument Parameters
Wavelength,

= 515.0 nm

Path length

= 10 mm

Absorbance vs Concentration
1
0.8

f(x) = 0.02x + 0
R = 1

0.6
Absorbance

0.4
0.2
0
0

10

15

20

25

30

35

40

45

Concentration

The standard calibration curve is obtained with the standard deviation of 0.999 and the
linear regression equation is:
y = 0.018x + 0.002
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 as follow:

50

i)

Unknown 1 (Absorbance, [A] = 0.274)

Y = 0.018x + 0.002
0.274 = 0.018x + 0.002
X = 15.11 ppm
ii)

Unknown 2 (Absorbance, [A] = 0.540)

Y = 0.018x + 0.002
0.540 = 0.018x + 0.002
X = 29.88 ppm
DISCUSSION
In the analysis of food colour experiment, there are 2 objective that need to be focus on.
The first objective was to determine max 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 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.
The sample was then been tested using the instruments. Two types of analysis were done,
which are, wavelength scanning and photometric scanning. max 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. Although the concentration
of unknown solutions has been obtained by the instrument, manual calculations still been done
for comparisons.
The data for absorbance and concentration was then been manually plotted. The equation
that been get by plotting the graph was Y = 0.018x + 0.002 and the standard deviation was 0.999.
The two unknown solution was calculated manually using the equation of the graph. The
concentration of unknown solution was been calculated:

Unknown 1:

15.11 ppm

Unknown 2:

29.88 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.
From the standard calibration graph that been manually plotted. The R2 value for the
Tartrazine is 0.999 is nearly to the true value, 1.000 according to the Beers Law. The Standard
Calibration Graph line is linear related to the Beers Law. So, that means, from this experiment
we know that Beers 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. The equation that been get by
plotting the graph was Y = 0.018x + 0.002 and the standard deviation was 0.999. The R2 value
for the Tartrazine is 0.999 is nearly to the true value, 1.000 according to the Beers Law.

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 mL

25 ppm
M1 V1

= M2 V2

(100ppm) (V1) = (25ppm) (50mL)


V1

= 12.5 mL

35 ppm
M1 V1

= M2 V2

(100ppm) (V1) = (35ppm) (50mL)


V1

= 17.5 mL

45 ppm
M1 V1

= M2 V2

(100ppm) (V1) = (45ppm) (50mL)


V1

= 22.5 mL

Pre Laboratory Question


1) State the Beers Lambert Law.
Beer Lambert law is represented by the formula
A= bc
Where A is absorbance and it does not have units, is the molar absorbtivity with units of L mol1

cm-1. b is the path length of the cuvette in which contain the sample. The unit is in centimetres.

c is the concentration of the compound in solution, expressed in M or mol L-1 .

2) What is the volume needed to prepare 50ppm of carmoisine from a 100ppm of carmoisine in
100ml volumetric flask?
50 ppm
M1 V1

= M2 V2

(100ppm) (V1) = (50ppm) (100mL)

V1

= 50 mL

Post Laboratory Questions


1) 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 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

2) Describe the function of wavelength scanning and photometric scanning.

The purpose of wavelength scanning are 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

i) Food Analysis, Third Edition, Kluwer Acedemic/Plenum Publishers, S. Suzanne Nielsen,


2003, New York, 2003
ii) Darrel D. Ebbing, Steven D. Gammon, General Chemistry Ninth Edition, Houghton
Company (2009).

Mifflin