TITLE

Determination of Riboflavin By Fluorescence Spectrophotometry

OBJECTIVE
To determine the amount of riboflavin in the energy drink.
ABSTRACT
In this experiment , we are going to determine the amount of riboflavin in the energy
drink. We are carried out the experiment involving the Fluorescence spectrophotometry. This
experiment is started by preparation of standard solution of riboflavin and sample. By analyzing
the standard solution of riboflavin, the data obtained can be the graph plotted. From this, we
calculate the amount of riboflavin in the sample. The amount of riboflavin that contain in the
energy drink for the sample 1 , 2 and 3 are 0.85mg ,0.85mg ,0.825 mg respectively.
INTRODUCTION
Riboflavin, vitamin B2 is a naturally fluorescent compound found in many foods such as
milk and eggs. It exhibits fluorescence in the visible region and which can be seen as greenyellow colour by the human eye. Riboflavin fluorescence is extremely sensitive to its
environment. It is sensitive to pH, presence of oxidizing species, and exposure to light. These
matrix effects can be difficult to correct for in quantitative analyses by direct measurement of
their influences on fluorescence intensity.
At room temperature, Riboflavin (Vitamin B2) can be promoted to various excited
vibrational levels within the first electronic excited state by absorption of UV and blue light.
Absorption is followed by vibrational relaxation, whereby a thermal population distribution of
excited molecules is reestablished such that they accumulate in the lowest vibrational levels of
the electronic excited state. The excited molecules is then relax to the various vibrational levels
of the ground state through a spontaneous emission process resulting in a green fluorescence.
Fluorescence spectroscopy is widely used in biomedical analyses because it has several
advantages over absorption spectrometry. Some of these advantages include:

1.

It is more selective since only a small subset of absorbing molecules fluoresce, and it has

two spectral variables; the excitation and emission wavelength,

2. It is more sensitive since the detector has only to sense the fluorescence
radiation,whereas in absorption measurement the detector must sense the small
absorption difference between blank and the sample. Detection methods that have a
signal level of zero when the analyte concentration is zero are known as zero
background techniques.
Fluorescence is sensitive to environmental factors, likes solvent polarity,hydrogen bonding,
temperature, pH, oxidation, and reduction. Riboflavin, for example, shows nearly constant
fluorescence from pH 4-8, but is nearly 100% quenched if the pH is raised to 10, or if the
molecule is reduced. Consequently, analytical fluorescence measurements are prone to errors
resulting from environmental influences on fluorescence intensity.

APPARATUS / MATERIAL

Volumetric flask
Acetic acid

PROCEDURE

Pipette
Energy drink

A. PREPARATION OF 5% ACETIC ACID SOLUTION.

1. 500 ml of 100 ppm acetic acid is prepared from 25 ml of acetic acid and top up
with deionised water.
B. PREPARATION OF STANDARD SOLUTION.
1. The 100 ppm acetic acid prepared was diluted to 10 ppm by adding 5ml of 100
ppm acetic acid to 50ml volumetric flask and top up with deionised water.
2. 5 ml of stock solution(10ppm) was pipette into a 50ml volumetric flask and
diluted with deionised water till calibration marked.
3. The standard was prepared as in table given below in 50 ml volumetric flask.

Concentration of standard (ppm)

0.02
0.04
0.06
0.08
0.10

Volume needed (ml)

1.0
2.0
3.0
4.0
5.0

4. For each concentration, the volume was pipette into a 50 ml volumetric flask as
stated in table above respectively.
C. PREPARATION OF SAMPLE
1. About 0.2 ml of energy drink is added to 50ml of volumetric flask and topup
with 5% of acetic acid till the calibration mark.
2. The step 1 is repeated 2 times.

RESULT AND DISCUSSION

The excitation and emission spectra of pure riboflavin and riboflavin in

the energy drink were measured using Fluorescence spectrometer in the spectral range of
370 to 594 nm. The result were presented in 2 graphs of intensity versus wavelength

Concentration of

standard (ppm)

i
n

which are excitation spectra and emission

spectra. From the emission spectra , it can

observe that riboflavin emitted in the

spectral range between 480nm to 594 nm

with the maximum wavelength at 270 nm.

The data obtained were tabulated below

0.02

y
8

.
5

0.04

8
1

4
.
9

0.06

1
2
3
7
.
8

0.08

9
3
1
6
.
7

0.1

8
3
8
7
.
9
7

The standard linear

calibration curve obtained of emission data

for the determination of riboflavin is
presented in Figure 1. It showed a good
linear relationship between the intensity and
concentrations of the standard solutions.

A graph of intensity against concentration

500
400
f(x) = 3743.25x + 14.83

300

intensity

intensity 200

Linear (intensity)

100
0
0 0.020.040.060.08 0.1 0.12
concentration (ppm)

From the graph plotted, the equation that obtained is y = 3743x + 14.83

So, we calculated the concentration of riboflavin in the energy drink using this equation
by substituting the value of intensity. And the dilution factor is 250.

Sample 1

y = 3743x + 14.83

142.93 = 3743x + 14.83

Sample 2

X= 0.034 ppm

y = 3743x + 14.83

[riboflavin] in 50 ml = 0.034 x 250

140.34 = 3743x + 14.83

=29.31 %

=8.5 ppm

X= 0.034 ppm

Mass of riboflavin= 8.5 mg/L x

[riboflavin] in 50 ml = 0.034 x 250

0.100 L

= 0.85mg
%weight = 0.85mg/2.9 mg x 100 %

=8.5 ppm

Mass of riboflavin= 8.5 mg/L x

0.100 L

= 0.85mg
%weight = 0.85mg/2.9 mg x 100 %

=29.31 %

Sample 3

y = 3743x + 14.83

138.41 = 3743x + 14.83

X= 0.033 ppm

[riboflavin] in 50 ml = 0.033 x 250

=8.25 ppm
Mass of riboflavin= 8.25 mg/L x 0.100 L

= 0.825 mg
%weight = 0.825 mg/2.9 mg x 100 %

=28.45 %

The major factor that effect the fluorescence intensity in this experiment is the

concentration of fluorescent solution. Normally, we control the concentration of solution for the
absorbance become smaller than 0.05. When the concentration is too high, the plot of intensity
against concentration will not be a linear. It is due to the intermolecular collision (both
fluorescent molecule and non-fluorescent molecule), transition into triplet excitation and
electronic transition.

Besides, Riboflavin fluorescence is very sensitive to pH environment, presence of

oxidizing species and exposure to light.

CONCLUSION

The amount of riboflavin in the energy drink for the sample 1 , 2 and 3 are 0.85mg ,
0.85mg ,0.825 mg respectively and the percentage weight are 29.31 %,29.31 % and 28.45
% respectively.

REFERENCES

1. Holler, Skoog&Crouch (2007).Principles of Instrumental Analysis (6th ed.). Thomson

Brooks/Cole
2. Fluorescence retrieved on May 20, 2015 from http://en.wikipedia.org/wiki/Fluorescence.
3. Basic concept in fluorescence retrieved on May 20, 2015 frm
http://www.olympusmicro.com/primer/techniques/fluorescence/fluorescenceintro.html