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CFB 40103 – Advance Food Analysis Practical 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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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

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) 2

CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

Figure 3 : One kind of Ultraviolet Visible Spectroscopy

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.

A∝C 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.

A∝b Combining Beer’s and Lambert’s expression, we have :

A ∝ bC Thus,

A

= єbc,

where є = molar absorptivity

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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

Figure 4 : UV-VIS Schematic Diagram

 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

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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

 Equipment Perkin Elmer UV-Vis Spectrophotmeter Lambda EZ210

 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 V 1 = M2 V 2 Where

to find the V1

M1 = concentration of carmoisine stock V1 = volume of carmoisine stock to be drawn M2 = concentration of carmoisine (diluted) V2 = volume of carmoisine (diluted)

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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

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.  Results Table 1 : Dilution Factors and Absorbance of Carmoisine

Blank Std 1 Std 2 Std 3 Std 4 Std 5 Unknown 1 Unknown 2

Concentration (ppm) 0 5 15 25 35 45

Absorbance [A] 0.000 0.193 0.403 0.564 0.911 1.321

10.778

0.285

19.740

0.527

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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

Figure 5 : Absorbance vs Concentration (Standard Calibration Graph for Carmoisine)

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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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

 Discussions Ultra-Visible determine

Spectrophotometer the

maximum

is

used

wavelength

in of

this

experiment

Carmoisine

to

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 8

CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

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. 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 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.

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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

The maximum wavelength in the experiment was obtained 510nm. There were no problems occurred while running the experiment.

 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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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

 Appendix Sample Calculations Preparation of Serial Dilutions •

5 ppm

M1 V 1

= M2 V 2

(100ppm) (V1) V1 •

= 2.5 mL

15 ppm

M1 V 1

= M2 V 2

(100ppm) (V1) V1 •

= 7.5 mL

= M2 V 2

(100ppm) (V1) V1

M1 V 1

= (15ppm) (50mL)

25 ppm

M1 V 1

•

= (5ppm) (50mL)

= (25ppm) (50mL)

= 12.5 mL

35 ppm = M2 V 2

(100ppm) (V1)

= (35ppm) (50mL) 11

CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

V1 •

= 17.5 mL

45 ppm

M1 V 1

= M2 V 2

(100ppm) (V1) V1

= (45ppm) (50mL)

= 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/UVVis/spectrum.htm

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CFB 40103 – Advance Food Analysis Practical 4 : UV-VIS Spectroscopy

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