Spectrofluorimetry

March 22, 2012

Minia University Faculty of Pharmacy Department of Analytical

1. Introduction
Absorption of UV/visible radiation causes transition of electrons from ground state (low energy) to excited state (high energy). As excited state is not stable, excess energy is lost by:
Collisional deactivation Emission of radiation (Photo Luminescence)

2. Molecular spectroscopy
The study of absorption or emission of light by molecules. Absorption and emission of light corresponds to absorption and emission of energy by the electrons of the molecule. The range of those energetic transitions is very wide, so that absorption and emission can be observed in any part of the electromagnetic spectrum. Hence, molecular spectroscopy can be divided in three different techniques, defined by the range of the spectrum observed:
UV-Visible Infrared - Nuclear magnetic resonance

2. Molecular spectroscopy
The energy levels of electrons in molecules are quantified. This is why each molecule can only absorb/emit discrete wavelength, corresponding to transitions from one level to another. Selection rules established through quantum mechanics calculation allow telling which transition is possible and which is not.

3. Spectrofluorimetry
The study of a specific mode of light emission by molecules called fluorescence. It can be used both for quantitative and qualitative analysis for research purpose, like the determination of the effect of different halides on the quenching of a molecule.

4. The origin of fluorescence

Subjected to excitation by light, a molecule (the solute, initially in the ground state S0), is instantaneously promoted to its first electronic excited state S1. The electrons and those of the neighboring solvent molecules re-equilibrate very quickly; however the position of the atomic nuclei remains identical to how they were in the fundamental state (this is the FranckCondon principle).

4. The origin of fluorescence

The solute/solvent cage system being out of equilibrium, will adopt a more stable conformation relative to the electronic excited state S1. Then, by the process of internal conversion, the molecule will rejoin 10-12 s, without emitting photons, the state V0 of level S1. If this level is compatible with the fundamental level the system can relax through a fluorescent step (10-11 to 10-8 s) during which the molecule returns to one of the vibrational states of the ground electronic state S0 with emission of photons as fluorescence.

SINGLET STATES

TRIPLET STATES

s2

VIBRATIONAL RELAXATION

T2 T1
INTERSYSTEM CROSSING PHOSPHORESCENCE INTERNAL CONVERSION INTERNAL CONVERSION

s1

FLUORESCENCE

Ground State

4. The origin of fluorescence

Singlet ground state: state in which electrons in a molecule are paired. [ ] Singlet excited state: state in which electrons are unpaired but of opposite spins. [ ] Triplet state: state in which unpaired electrons of same spin are present. [ ] Excitation process: absorption of energy or light followed by conversion from ground state to excite state. Relaxation process: process by which atom or molecule losses energy & returns to ground state.

5. Photoluminescence
Light without heat or cold light Basically of 2 types
Fluorescence: part of energy is lost due to vibrational transitions and remaining energy is emitted as UV/visible radiation of longer wavelength than incident light. Phosphorescence: under favorable conditions, excited singlet state undergo transition to triplet state. Emission of radiation when e- undergo transition from triplet state to ground state.

6. Classification of fluorescence
Based on the wavelength of emitted radiation when compared to absorbed radiation
Stokes fluorescence: wavelength of emitted radiation is longer than absorbed radiation Anti-stokes fluorescence: wavelength of emitted radiation is shorter than absorbed radiation. Resonance fluorescence: wavelength of emitted radiation is equal to that of absorbed radiation.

7. Factors affecting fluorescence

The common factors affecting the fluorescence are as follows:
Temperature pH Dissolved oxygen Solvent

7.1. Temperature
A rise in temperature is almost always accompanied by a decrease in fluorescence. The change in temperature causes the viscosity of the medium to change which in turn changes the number of collisions of the molecules of the fluorophore with solvent molecules. The increase in the number of collisions between molecules in turn increases the probability for deactivation by internal conversion and vibrational relaxation.

7.2. pH
Relatively small changes in pH can sometimes cause substantial changes in the fluorescence intensity and spectral characteristics of fluorescence.
For example, serotonin shows a shift in fluorescence emission maximum from 330 nm at neutral pH to 550 nm in strong acid without any change in the absorption spectrum.

7.2. pH
The changes in pH of the medium change the degree of ionization of the acidic/basic functional groups. This in turn may affect the extent of conjugation or the aromaticity of the molecule which affects its fluorescence.
For example, aniline shows fluorescence while in acid solution it does not show fluorescence due to the formation of anilinium ion.

Therefore, pH control is essential while working with such molecules and suitable buffers should be employed for the purpose.

7.3. Dissolved oxygen

The paramagnetic substances like dissolved oxygen and many transition metals with unpaired electrons dramatically decrease fluorescence and cause interference in fluorimetric determinations. The paramagnetic nature of molecular oxygen promotes intersystem crossing from singlet to triplet states in other molecules. The longer lifetimes of the triplet states increases the opportunity for radiationless deactivation to occur.

7.3. Dissolved oxygen

Presence of dissolved oxygen influences phosphorescence too and causes a large decrease in the phosphorescence intensity. It is due to the fact that oxygen which is in triplet state at the ground state gets the energy from an electron in the triplet state and gets excited. This is actually the oxygen emission and not the phosphorescence. Therefore, it is advisable to make phosphorescence measurement in the absence of dissolved oxygen.

7.4. Solvent
The changes in the polarity or hydrogen bonding ability of the solvent may also significantly affect the fluorescent behavior of the analyte. The difference in the effect of solvent on the fluorescence is attributed to the difference in their ability to stabilize the ground and excited states of the fluorescent molecule. Besides solvent polarity, solvent viscosity and solvents with heavy atoms also affect fluorescence and phosphorescence.

7.4. Solvent
Increased viscosity increases fluorescence as the deactivation due to collisions is lowered. A higher fluorescence is observed when the solvents do not contain heavy atoms while phosphorescence increases due to the presence of heavy atoms in the solvent.

8. Photoluminescence and structure

Compounds with fused ring are found to be especially fluorescent, and the extent of fluorescence is found to be directly proportional to the number of rings in the molecule The structural rigidity in a molecule favors fluorescence Aliphatic and alicyclic carbonyl compounds or highly conjugated double bond structures also show fluorescence.

8. Photoluminescence and structure

The fluorescence observed with rigid cyclic molecules with pi-bonds is found to be enhanced by electron donating groups e.g., NH2, OR, OH and OCH3, The electron withdrawing groups such as COOH, NO2, N=N and Br, I and CH2COOH tend to reduce it. On the other hand the non-rigid molecules do not fluoresce much, as these rapidly lose the absorbed energy through non-radiative means like, vibrational relaxation or even degradation.

9. Instrumentation for fluorescence

The essential components of an instrument used to measure fluorescence of the sample are:
Excitation light sources Filters or Monochromators Sample holder Detector Readout device

9. Instrumentation for fluorescence

Source of light
Mercury vapor lamp: Hg vapor in high pressure (8 atm) gives intense lines on continuous background above 350nm. Xenon arc lamp: gives more intense radiation. Tungsten lamp: used if excitation has to be done in Vis. region.

9. Instrumentation for fluorescence

Filters and monochromators
In fluorimeter 10 filter (absorb Vis. radiation and transmit UV radiation) and 20 filter (absorb UV radiation and transmit Vis. radiation) are present. In spectrofluorometers, excitation monochromators and emission monochromator are present.

Sample cells
Sample cells are cylindrical or polyhedral made up of color corrected fused glass & path length normally 10 mm to 1 cm.

9. Instrumentation for fluorescence

Detectors
Photo voltaic cell Photo tubes Photo multiplier tubes

10. Advantages of fluorimetry

More sensitive when compared to other absorption techniques. Concentrations as low as g/ml or ng/ml can be determined. As both excitation & emission wave lengths are characteristic it is more specific than absorption methods.

11. Applications of fluorimetry

Determination of Organic substances
Plant pigments, steroids, proteins, etc. can be determined at low concentrations. Generally used to carry out qualitative as well as quantitative analysis for a great aromatic compounds present in cigarette smoking, air pollutant concentrates & automobile exhausts.

Determination of inorganic substances Extensively used in the field of nuclear research for the determination of uranium salts. Determination of vitamin B1 (thiamine) in food samples like meat cereals etc.

11. Applications of fluorimetry

Determination of Vitamin B2 (riboflavin). This method is generally used to measure the amount of impurities present in the sample. Most important applications are found in the analyses of food products, pharmaceuticals, clinical samples and natural products. Fluorescent indicators:
Intensity and color of the fluorescence of many substances depend upon the pH of solutions. These are called as fluorescent indicators and are generally used in acid base titrations. Eosin : pH 3.0-4.0 colorless to green Fluorescein : pH 4.0-6.0 colorless to green

 Ahmed Zaghloul Helbawi Eslam Ahmed Abdel Azeem Ahmed Mohamed Harb Ahmed Abdel Naser Mohamed Ahmed Ali Desoky Ahmed Ali Mohamed Eslam Salah Ibrahim Gaber Mohamed Gaber Ahmed Eid Ibrahim Ahmed Lamie Fekry Ahmed Gamal El Deen Ahmed Adel Abdel Gaber Ahmed Mostafa Yehya Ahmed Mohamed Hosny