EXPERIMENT 3

1. What is the purpose /objective of this experiment?

To analysis of vitamin B2, riboflavin, by measuring its native fluorescence.

2. What are the Sources of Luminescence? How we classify the luminescence?

Luminescence can be classifieds according to the source of excitation into:
- Photo-luminescence: deactivation takes place after excitation with photons.
- Radio-luminescence: ground state molecules are excited by collisions with high energy particles.
- Chemo-luminescence: ground state molecules are excited by certain chemical reactions.

3. What are the Characteristics of the following:

- Fluorescence: is short-lived with luminescence ending almost immediately.
- Phosphorescence: involves change in electron spin and may endure for several seconds.
- Photo-luminescent: in most cases, radiation tends to be at longer wavelengths than excitation
radiation.
- Chemiluminescence: is based on an excited species formed by a chemical reaction.

4. Types of Fluorescence/phosphorescence
- Resonance radiation (or fluorescence); absorbed radiation is reemitted without alteration.
- More often, molecular fluorescence (phosphorescence) occurs as bands centered at wavelengths
longer than resonance line.

5. Define the stokes shift.

- The shift to longer wavelengths in the resonance radiation.

6. How to calculate the molecular multiplicity, M?

- M = 2S + 1
- where S is the spin quantum number of the molecule
- M = sum of the net spin of the electrons in the molecule

7. Explain why most of the organic molecules have S = 0 ?

- Because the molecules have even number of electrons, therefore, the ground state must have all
electrons paired, so S = 0.
8. Why molecules in the ground state mostly have a singlet state?
- Because S = zero and (M = 2 * 0 + 1 = 1), and S0 . S1 and S2 for first and second excited states.
9. Why some molecules in the excited states have Triplet state, T1 ?
- Because in the excited state, one electron may reverse its spin, so:
! !
𝑆 = #"$ + #"$ = 1 , so when we calculate 𝑀 = 2 ∗ (1 ) + 1 = 3 = Triplet state = T1

10. Why a molecule in the ground state cannot have a triplet state?
- Because the molecules in the ground state have even number of electrons, thus the spins of all
electrons are paired, S=0.

11. Which molecules can have a doublet state?

- The molecules that have one unpaired electron. Examples are the organic free radicals.

12. Draw the spin orientation of the singlet, doublet, and triplet states.
Using the above figures:

13. Explain the Singlet State.

- Electron spins are paired, no splitting of energy level. May be ground or excited state.

14. Explain the doublet State.

- Free radical (due to odd electron).

15. Explain the Triplet State.

- One electron excited to higher energy state, spin becomes unpaired (parallel).

16. NOTE: A change in electron spin is technically a forbidden process.

17. What does the term ‘Forbidden’ means?

- Forbidden process (according to quantum mechanics) means unlikely, but (not absolutely cannot
happen).

18. In which state the allowed absorption process occurs?

- The allowed absorption process will result in a singlet state.

19. In which states the molecules are paramagnetic and diamagnetic?

- In the T excited state, the molecule is paramagnetic
- In the singlet state, the molecule is diamagnetic.

20. Which transitions of electrons between the states are less likely to occurs?
 - S → T transitions or reverse, are less probable than the S → S transitions.

21. Which transitions of electrons between the states make the absorption peaks less sensitive?
- The S → T transitions are less sensitive than the S → S transitions.

22. What are average lifetime of the singlet and the triplet excited states? And which one is longer?
- The average lifetime of the Triplet excited state is (10-4 sec) which is longer than the singlet excited
state (10-5 - 10-8 sec).

23. Explain how the phosphorescence process can occurs?

- When an excited T state can be populated from an excited S state of certain molecules, a
phosphorescence process will be the result.

24. Explain the differences between the S and T states. Sensitivity, lifetime, para- or dia-, which one less
probable to occur.

25. Energy of a Molecule (Jablonski energy-level diagram):

26. Compare between the different types of emission:

- Fluorescence: return from excited S state to ground state; does not require change in spin orientation
(more common of relaxation).
- Phosphorescence: return from T excited state to a ground state; electron requires change in spin
orientation.

27. Compare the emissive rates between the fluorescence and the phosphorescence.
- Fluorescence emissive rates are: (10-10-10-7 s) and are several orders of magnitude faster than that of
phosphorescence (10-6-10 s).

28. Explain Excitation in the Fluorescence process:

- At room temperature, everything starts out at lowest vibrational energy level of the ground state.
- Suppose molecules is illuminated with light at resonance frequency.
- Light is absorbed; for diluted sample, Beer Lambert law applies A=εlc
- Excitation following light absorption, a chromophore is excited to some
higher vibrational energy level S1 or S2.
- The absorption process takes place on a time scale (10-15s) much faster
than that of molecular vibration vertical transition (Frank-Condon).
29. Explain the Mirror image rule.
- Vibrational levels in the excited states and ground states are similar.
- An absorption spectrum reflects the vibrational levels of the electronically
excited state.
- An emission spectrum reflects the vibrational levels of the electronic ground
state.
- Fluorescence emission spectrum is mirror image of absorption spectrum.
30. Compare the Internal conversion vs. fluorescence emission.
- As electronic energy increases, the energy levels grow closely spaced.
- It is more likely that there will be overlap between the high vibrational energy
levels of Sn-1 and low vibrational energy levels of Sn.
- This overlap makes transition between states highly probable.
- Internal conversion is a transition occurring between states of the same
multiplicity and it takes place at a time scale of 10-12 s (faster than that of
fluorescence process).
- The energy gap between S1 and S0 is significantly larger than that between
other adjacent states.
- S1 lifetime is longer radiative emission can compete effectively with non-
radiative emission.
31. Define the stokes's shift.
- The Stokes’s shift is the gap between the maximum of the first absorption band
and the maximum of the fluorescence spectrum.

- loss of vibrational energy in the excited state as heat by collision with solvent
32. Example: 7-amino-4-methylcoumarin (AMC):
33. Example: Fluorophores (Fluorescein):

34. Example: (Ethidium bromide bound to DNA):

35. Explain the desighns of the Fluorometers instruments.

- The source beam is split near the source into a reference beam and a sample beam.
- The reference beam is attenuated by the aperture disk so that its
intensity is roughly the same as the fluorescence intensity.
- Both beams pass through the primary filter, with the reference beam
then being reflected to the reference photomultiplier tube.
- The sample beam is focused on the sample by a pair of lenses and
causes emission of fluorescent radiation.
- The emitted radiation passes through a second filter and then is focused
on the second photomultiplier tube.
- The electrical outputs from the two detectors are fed into a solid state
comparator, which computes the ratio of the sample to reference
intensities; this ratio serves as the analytical parameter.
- Nearly all fluorometers (spectrofluorometers) are double-beam systems.

36. Write down the equations that included in this experiment.

Where:

- I is the transmitted light intensity,

- I0 is the incident intensity,
- ε is the molar absorptivity at a given wavelength in units of L*mol-
1*cm-1

- b is the cell path in centimeters, and

- c is the concentration in moles per liter.
- F, is fluorescence intensity
- Φ fluorescence quantum yield
- k is a proportionality constant
 37. Experimental Part.
- Riboflavin (A) and the two most predominant irradiation decomposition
- products, lumiflavin (B) and lumichrome (C).

¨Very Toxic ¨Toxic ¨Harmful ¨Irritant ¨ Corrosive

¨Flammable ¨Explosive

Open bench only ¨ Good Chemical Laboratory Practice x

Fume cupboard needed ¨ Eye protection x
Local exhaust ventilation ¨ Gloves x
Total enclosure ¨ Other ¨

Experimental Method
- First, in this experiment we are going to calculate the concentrations of Riboflavin samples at ppm level,
therefore, carful cleaning and preparation is required.
- The 100-ppm stock standard solution was obtained from the lab supervisor, then it was allowed to reach
the room temperature.
- Using a 50 mL volumetric flasks, 2,3,4,5, and 6 mL of the stock solution were diluted with deionized
water.
- Then the samples run on the Fluorometer, and the data were saved.
- The optimum excitation wavelength for riboflavin was determined. The absorption spectrum was used to
approximate the best excitation wavelength if there is no prior information were available.
 Results and discussion
The mass of Vitamin B-2 sample = 1.500 g
The solution volume = 100.0 mL
Initial concentration of Riboflavin = 100 ppm

Concentrations: For instance, at 2 mL

M1V1 = M2V2
100 * 2 = M2 * 50.0
M2 = 4 ppm
"
At 2 mL, M2 = 100 ∗ #$.$ = 4 𝑝𝑝𝑚
&
At 3 mL, M2 = 100 ∗ #$.$ = 6 𝑝𝑝𝑚
'
At 4 mL, M2 = 100 ∗ #$.$ = 8 𝑝𝑝𝑚
#
At 5 mL, M2 = 100 ∗ #$.$ = 10 𝑝𝑝𝑚
(
At 6 mL, M2 = 100 ∗ #$.$ = 12 𝑝𝑝𝑚

Table 1: The standard samples, their maximum wavelengths and the fluorescences:
Standard Vi (mL) Vf (mL) [Riboflavin] in ppm Fluorescences

1 2 50.0 4 16326

2 3 50.0 6 24355

3 4 50.0 8 41435

4 5 50.0 10 50176

5 6 50.0 12 60848

Riboflavin - - 4.670 19505

 Absorption Spectrum
70000

60000

50000 Wavelength nm.

Absorbance

40000 2ml
3ml
30000
4ml
20000
5ml
10000
6ml
0 Unk
62

1038
1099
1160
1

123
184
245
306
367
428
489
550
611
672
733
794
855
916
977
-10000
Wavelength (nm)

Figure 1: The absorbance Vs. The wavelengths.

Maximum Volume Fluorescence
wavelength (mL) (Max)
533 2 16326
533 3 24355
533 4 41435
533 5 50176
533 6 60848
533 Riboflavin 19505

70000
y = 5743.3x - 7318
60000 R² = 0.9875
50000
[Fluorscence]

40000

30000

20000

10000

0
0 2 4 6 8 10 12 14
[Riboflavin]

Figure 2: The calibration curve of Vitamin B-2 concentration and Fluorescences.

The regression equation is: y= 5743.3x – 7318
At y = 19505, 19505 = 5743.3𝑥 – 7318
19505 + 7318
𝑋= = 4.67 𝑝𝑝𝑚
5743.3
!$$
In 1L, the mass is = 4.670 𝑝𝑝𝑚 ∗ #!$$$$ = 0.467 𝑝𝑝𝑚

= 0.000467 g/L
= 0.000467 g
$.$$$'()
% mass in the tablet = # $ ∗ 100 = 0.0311%
!.#$$