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 solution, the

cuvettes was inserted in the sample

compartment. The sides of the cuvettes

were wiped clean and the clean surface

cannot touch. The wavelength scan is done

and the λmax 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 the

standard calibration graph is produced.

RESULT

A) The Preparation of Serial Dilutions

STOCK DILUTED

Concentration of Carmoisine stock (ppm)

M1

Volume of Carmoisine stock (ml)

V1

Concentration of Carmoisine stock (ppm)

M2

Volume of Carmoisine stock

(ml)

V2

1 100 (100) V1 = (5)(50) V1 = (5)(50)

100 = 2.5 ml

5 50

2 100 (100) V1 = (15)(50) V1 = (15)(50)

100 = 7.5 ml

15 50

3 100 (100) V1 = (25)(50) V1 = (25)(50)

100 = 12.5 ml

25 50

4 100 (100) V1 = (35)(50) V1 = (35)(50)

100 = 17.5 ml

35 50

5 100 (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

(2) Result for Wavelength Scan

Sample used = Carmoisine stock

Concentration of the sample = 45 ppm

λ max obtained = 515.0 nm

C. Photometric Scan

(1) Instrument Parameters

Wavelength, λ = 515.0 nm

Path length = 10 mm

Standard no. Concentration (ppm) Absorbance

Blank 0 0.000

Std 1 5 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

0 5 10 15 20 25 30 35 40 45 500

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

f(x) = 0.0184827397260274 x + 0.00210958904109582R² = 0.999906806163134

Absorbance vs Concentration

Concentration

Abso

rban

ce

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:

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 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. 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 Beer’s Law. 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. 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 Beer’s 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 Beer’s 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

mol-1 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 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

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 Mifflin

Company (2009).