report uv-vis yaya

CFB 40103 – Advance Food AnalysisPractical 4 : UV-VIS Spectroscopy

Objective

1. To determine λmax for Carmoisine sample (wavelength scan)

2. The prepare a serial dilution and generate a standard calibration graph for

sample quantitation (photometric scan)

Introduction

Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-VIS)

involves the spectroscopy of photons and spectrophotometry. It uses light in visible and

adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this region of energy

space molecules undergo electronic transitions. Electromagnetic radiation in th 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.

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Figure 1 : Electromagnetic Spectrum

Figure 2 : Visible Spectrum

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 :

A = - log (%T)

Figure 3 : One kind of Ultraviolet Visible Spectroscopy

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Like Colorimeter and Atomic Absorption Spectroscopy (AAS), UV-VIS also applies Beer-

Lambert Law, which is the combination of Beer’s Law and Lambert’s Law. Beer’s Law is

defined as the absorbance (A) is directly proportional to concentration of solution (C)

when a beam of monochromatic light is passed through a solution of constant length.

Lambert’s Law is defined as the absorbance (A) is directly proportional to thickness of

solution (b) when beam of monochromatic light is passed through a solution of constant

concentration.

Combining Beer’s and Lambert’s expression, we have :

Thus,

Figure 4 : UV-VIS Schematic Diagram

A C

A b

A bC

A = єbc,

where є = molar

absorptivity

3


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

Equipment

Perkin Elmer UV-Vis Spectrophotmeter Lambda EZ210

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Methods

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 λmax 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.

9. Work station was cleaned properly before leaving the laboratory.

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Results

Table 1 : Dilution Factors and Absorbance of Carmoisine

Concentration (ppm) Absorbance [A]Blank 0 0.000Std 1 5 0.193Std 2 15 0.403Std 3 25 0.564Std 4 35 0.911Std 5 45 1.321

Unknown 1 10.778 0.285Unknown 2 19.740 0.527

0 5 10 15 20 25 30 35 40 45 500.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

f(x) = 0.027467397260274 x − 0.00690410958904109R² = 0.976427141469739

Absorbance [A] vs Concentration (ppm)

Concentration (ppm)

Abso

rban

ce [A

]

Figure 5 : Absorbance vs Concentration (Standard Calibration Graph for

Carmoisine)

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The standard calibration curve is obtained with the standard deviation of 0.976

and the linear regression equation is :

y = 0.027x – 0.006

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 :

Unknown 1 (Absorbance, [A] = 0.285)

y = 0.027x – 0.006

0.285 = 0.027x – 0.006

x = 10.778 ppm

Unknown 2 (Absorbance, [A] = 0.527)

y = 0.027x – 0.006

0.527 = 0.027x – 0.006

x = 19.740 ppm

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Discussions

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.

In this experiment, the stock solution of 100ppm Carmoisine 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 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. 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 λmax 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.

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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 re-plotted manually

to obtain the equation of linear regression using Microsoft Office Excel software. The

equation obtained with standard deviation of 0.976 is :

y = 0.027x – 0.006

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 510nm.

There were no problems occurred while running the experiment.

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Conclusion

The experiment was successfully done and the objectives of the experiment are

achieved. The concentrations of two unknown solutions had been calculated to be

10.778 ppm and 19.740 respectively. The maximum wavelength, λmax for Carmoisine

sample is 510nm.

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Appendix

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)

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V1 = 22.5 mL

Data of Results

(attached in the end of the report)

References

Books

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

Nielsen, 2003, New York, 2003

Lecture Notes

The Influences of Color in the Acceptance of Jellies, Nadiah bt Mohd Kahar,

UniKL MICET, 2007

Websites

http://elchem.kaist.ac.kr/vt/chem-ed/spec/uv-vis/uv-vis.htm

http://en.wikipedia.org/wiki/Ultraviolet-visible_spectroscopy

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

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http://en.wikipedia.org/wiki/Ultraviolet-visible_spectroscopy

http://elchem.kaist.ac.kr/vt/chem-ed/spec/uv-vis/uv-vis.htm

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