Fluorescence Spectros

AS245: Bachelor in Science Applied Chemistry
Title: Analysis of Riboflavin in Energy Drink

Course: Spectrochemical Methods of Analysis
(CHM580)
Name: Mohamad Nor Amirul Azhar b. Kamis
Matrix no:
2014647344 (AS245 3S)
Date of experiment: 15th April 2015

Date of submission: 29th May 2015
Group members: 1. Nurnailah bt. Noorazlan
2. Nor Amirah bt. Ahmad Azuan
3. Noramira bt. Saad
It is my responsibility as a student of UiTM to adhere
to truthfulness and avoid dishonesty, fraud or deceit
of any type in connection with write up and conduct
of this experiment.
Signature:__________
Date:
TITLE:
Determination of amount riboflavin in energy drinks samples.
ABSTRACT:
Sample used in this experiment is the energy drinks (livita) that was diluted into
smaller concentration with dilution factor of 250, so that the intensity will be in the
range of standard solutions. Fluorescence Emission Spectroscopy was used to
analysed the analyte of riboflavin in the sample to know the concentration of the
riboflavin in the sample. The sample has been triplicate to ensure the accuracy and
getting more accurate data. The average concentration of riboflavin in the triplicate
sample is 9.725ppm (actual concentration) with relative standard deviation of 10.66
with average amount of riboflavin of 0.4863mg.
INTRODUCTION:
Riboflavin with molecular formula of C 17H20N4O6 is also known as vitamin B 2 is a part
of vitamin B group. It was formerly known as vitamin G with yellowish orange solid
substance that poorly soluble in water. The person who took this vitamin as
supplement can be visually observed from the colour the urine that will be yellow in
colour. Riboflavin can be obtained from many sources such as milk, liver, kidneys,
yeast, mushrooms and almonds. For this experiment, the riboflavin is get from the
energy drinks. RDA recommends riboflavin intake for adult men and women with
1.3mg/day and 1.1mg/day respectively. Infants and children are recommended to
take riboflavin in lighter amount of 0.3-0.4mg/day for infants and 0.6-0.9mg/day for
children. Too much riboflavin will cause stomatitis including painful red tongue with
sore throat, chapped and fissured lips (cheilosis) and inflammation of the corners of
the mouth (angular stomatitis).
OBJECTIVES:
1. To determine the concentration of riboflavin in energy drinks.
EXPERIMENTAL:
A. Preparation of reagents:
1. Preparation of 5% acetic acid solution:
a. 25mL of concentrated acetic acid solution was pipetted into a 500mL
volumetric flask and was diluted until the calibration mark by using deionised water.
B. Preparation of standards solutions:
1. Preparation of 100ppm riboflavin stock solution:
a. 10mg of riboflavin was dissolved by using 5% acetic acid solution.
b. The solution then was transferred into a 100mL volumetric flask and
was marked up by using 5% acetic acid solution.
2. Preparation of 10ppm riboflavin solution from 100ppm riboflavin stock
solution:
C1V1 = C2V2
100V1 = 10(50)
V1 = 5mL
a. 5mL of 100ppm riboflavin stock solution was pipetted into a 50mL
volumetric flask and was marked up by using 5% acetic acid solution.
3. Preparation of standard solutions:
Riboflavin working solution = 10ppm
Standard
Concentration (ppm)
Volume of 10ppm
riboflavin working
Blank
1
2
3
4
5
0
0.02
0.04
0.06
0.08
0.10
solution pipetted (mL)

0
0.1
0.2
0.3
0.4
0.5
a. The volume of 10ppm riboflavin working solution was pipetted into

each standard of 50mL volumetric flasks based on the table above.
b. The 5% acetic acid solution was used to mark up the solution until the
calibration mark of 50mL.
Sample calculation of standard solution 1:
C1V1 = C2V2
10V1 = 0.02(50)
V1 = 0.1mL
C. Preparation of sample:
a. 0.2mL of sample was pipetted into a 50mL volumetric flask and was
marked up until the calibration mark by using 5% acetic acid solution.
Calculation of dilution factor for sample:
50
DF = 0.2
= 250
RESULT:
a. Excitation data at 451.62nm (max)
Blank (0.00ppm)
Standard 0.02ppm
Standard 0.04ppm
Standard 0.06ppm
Standard 0.08ppm
Standard 0.10ppm
Sample A
Sample B
Sample C
Intensity
18.13
110.32
163.22
238.05
310.47
370.89
158.40
178.56
150.58
b. Emission data at 530.51nm (max)

Blank (0.00ppm)
Standard 0.02ppm
Standard 0.04ppm
Standard 0.06ppm
Standard 0.08ppm
Standard 0.10ppm
Sample A
Sample B
Sample C
Intensity
15.62
108.42
161.55
236.14
305.44
367.89
156.35
176.61
148.82
Graph of emission intensity versus concentration

400
350
f(x) = 3467.14x + 25.82

R = 1
300
250
Intensity
200
150
100
50
0
0
0.02
0.04
0.06
0.08
0.1
0.12
Concentration (ppm)
Sample calculation of concentration of sample: sample A intensity = 156.35

y = 3467.1x + 25.82
156.35 = 3467.1x + 25.82
x = 0.0376ppm
Intensity
156.35
176.61
148.82
Sample A
Sample B
Sample C
Average concentration of sample =
Concentration (ppm)
0.0376
0.0435
0.0355
0.0376+0.0435+0.0355
3
= 0.0389ppm
*See appendix for graph that show how the concentration of sample is obtained.
Standard deviation =
1
( x imean)2
N 1 i
1
2
2
2
[( 0.03760.0389 ) + ( 0.04350.0389 ) + ( 0.03550.0389 ) ]
31
= 4.1479
10-3
Relative standard devation, %RSD
standard deviation
mean
4.1479 103
0.0389
100%
100%
= 10.66
Actual concentration of sample (average)
= 0.0389ppm 250 (dilution factor)

= 9.725ppm
mg
Average amount of riboflavin in sample = 9.725 L
0.05L
= 0.4863mg
DISCUSSION:
The process of fluorescence begin with the absorption of UV radiation by molecule of
analyte, and the molecule will be excited to the higher electronic state from the
ground state, continue with vibrational relaxation and internal conversion between
same multiplicity and finally falls down back to the ground state while emitting the
fluorescence. For excitation maximum wavelength determination, the emission

wavelength was fixed while the excitation wavelength was varied in a range. From
the experiment, the maximum wavelength for excitation lies on 451.62nm. When
performing the emission analysis, the excitation wavelength was fixed and the
emission wavelength was varied in range. The maximum wavelength of emission is
530.51nm that is at longer wavelength compared to excitation wavelength. As all
knowing that energy is inversely proportional to the wavelength, it is proven that
emission of fluorescence occur at lower energy as compared to absorption of
radiation by referring to the maximum wavelength of excitation and emission. The
maximum wavelength is also as the qualitative analysis for specific analyte. Each
analyte has its own specific maximum wavelength to excite and emit the radiation in
order to determine them.
From the graph, it is shown a good standard solution preparation due to nearer of
the value of R2 to 1. By using the equation of the straight line, the concentration of
each sample can be determined. The relative standard deviation percentage
showing the value of 10.66 that indicates low precision between the triplicate sample
intensity values. The good precision will bring the %RSD nearer to zero. For this
experiment, the improvement is a need for the sample preparation. The precaution
such as washing the apparatus before working is compulsory to avoid the
contamination. The average amount of riboflavin in the triplicate sample is 0.4863mg
and it is safe to be consumed by adults but it is not suitable for infants.
CONCLUSION:
The actual average concentration of riboflavin in the livita energy drink sample is
9.725ppm. The average amount of riboflavin in the triplicate sample is equal to
0.4863mg.
REFERENCES:
1. Riboflavin, Retrieved April 26, from http://en.m.wikipedia.org/wiki/Riboflavin
2. F. J. Holler, D. A. Skoog, S. R. Crouch (2007), Principles of Instrumental
Analysis 6th ed.), United States, Brooks/Cole Cengage Learning.

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AS245: Bachelor in Science Applied Chemistry

Title: Analysis of Riboflavin in Energy Drink

2014647344 (AS245 3S)

Date of experiment: 15th April 2015

Determination of amount riboflavin in energy drinks samples.

solution pipetted (mL)

a. The volume of 10ppm riboflavin working solution was pipetted into

b. Emission data at 530.51nm (max)

Graph of emission intensity versus concentration

f(x) = 3467.14x + 25.82

Sample calculation of concentration of sample: sample A intensity = 156.35

Average concentration of sample =

Relative standard devation, %RSD

Actual concentration of sample (average)

= 0.0389ppm 250 (dilution factor)

fluorescence. For excitation maximum wavelength determination, the emission

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