RESULTS

Riboflavin standard (ppm) Fluorescence intensity (a.u.)

Wavelength (nm)
1
108.643
525.07
2
152.077
525.97
3
169.908
522.98
4
213.146
525.07
5
256.359
525.07
Table 1.1 : Fluorescence intensity for different concentration of riboflavin
standard.

Figure 1.1: Graph of fluorescence intensity versus concentration of riboflavin standards

Unknown sample
Fluorescence intensity (a.u)
2.5 times dilution
753.451
25 times dilution
233.400
Table 1.2 : The fluorescence intensity for unknown sample

CALCULATIONS
1. Concentration of Riboflavin in the unknown sample
From the graph of fluorescence intensity against concentration of riboflavin standards, a
linear equation was obtained, and used to calculate the concentration of riboflavin in the
unknown sample.
y = 35.65x + 73.076
Substitute y with the fluorescence intensity of unknown sample (25 times dilution)
233.400 = 35.65x + 73.076
x=( 233.40073.076)/35.65
x=4.4972 ppm

The actual concentration of riboflavin in the unknown sample

= 4.4972 ppm dilution factor
= 4.4972 ppm 25
= 112.43 ppm
2. Concentration (g) of riboflavin in sample

112.43

mg 10 g

L
L

112430

g
L

DISCUSSION
In this experiment, fluorescence spectroscopy was used to obtain
the wavelength of excitation and emission of the riboflavin sample and
also the concentration of riboflavin in unknown sample.

Figure 1.2 : Cary Eclipse Fluorescence Spectrophotometer

For this experiment, Cary Eclipse Fluorescence Spectrophotometer was used.
Fluorescence spectroscopy is widely used in biomedical analyses because it has several
advantages over absorption spectrometry. Some of these advantages are firstly, it is more
selective since only a small subset of absorbing molecules fluoresce, and it
has two spectral variables; the excitation and emission wavelength. Next is, 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. Furthermore, fluorescence is generally sensitive to environmental
factors, such as 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.
From the fluorescence spectrum, it shown that the intensity is directly proportional to the
concentration of sample. The reading for unknown sample was out of the range of the
standard, thus after another dilutions, 10 times dilution, the reading was in the range of the
standard.

Figure 1.3 : Five series of riboflavin standard solution and an unknown sample.

In this experiment, a calibration curve was used to study concentration (in units of ppm)
based on the fluorescence result. Therefore, we took readings from five different
concentrations of riboflavin: 1, 2, 3, 4, and 5 ppm and we get calibration curve as shown in
Figure 1.1 above. From Figure 1.1, the correlation coefficient (R2) value of the graph was
0.985 which indicates a positive correlation and practically linear, but not perfectly. Besides
that, from the graph, we also obtained a linear equation of y=35.65 x+ 73.076 . This
equation was used to determine the concentration of riboflavin the unknown sample by using
the fluorescence intensity for the unknown sample. The concentration of riboflavin in the
unknown sample was 112.43 ppm. However, the concentration calculated was out of range as
the concentration range of the unknown solution was 5 to 15 ppm. This might because of
some error during the experiment.
Based on the results of the experiment, some precaution steps should be taken to
minimize the error during experiment. Firstly, all the apparatus need to be ensure that they
were cleaned before use. Then, the apparatus need to be washed with an appropriate soap or
detergent, and rinse thoroughly with distilled or deionized water. Other than that, do not
pipette directly from a stock bottle as it will contaminate the solution and error occurs and the
pipette should be rinsed with the solution before use. Volumetric flask also should be allowed
to dry by evaporation. When using the fluorescence spectrophotometer, ensure that the
sample cell to be cleaned from the previous solution as it will causes the concentration
become different. Lastly, all the riboflavin should dissolved completely in acetic acid as
riboflavin must reacted with acetic acid to become fluorescent.

CONCLUSION
As for conclusion, this experiment was done to study and observe fluorescence,
spectrophotometry, and riboflavin. A calibration graph was conducted to show the linear
relationship between fluorescence and the concentration of a riboflavin sample. Thus, from
the results obtained, fluorescence spectrophotometry is a suitable method for determining the
wavelength of emission and excitation of riboflavin. Besides that, by using this method, the
concentration of riboflavin in unknown sample was determined. The concentration of
riboflavin in unknown sample is 112.43 ppm.

REFERENCES
1. Lakowicz, J. R. (Ed.). (2013). Principles Of Fluorescence Spectroscopy. Springer Science
& Business Media.
2. Kohl, J., (2014), Determination Of Riboflavin In Multivitamin, Fluorescence
Spectroscopy, Revised 10/16/14.