Fluorometry (fluorescence and Phosphorescence Study), Slides of Pharmaceutical Analysis

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Flourometry

Prepared by:

Runa Akter

Lecturer, Uap

Fluoresecnce

Fluorescence is a spectrochemical method of analysis where the molecules of the analyte are excite by irradiation at a certain wavelength and emit radiation of a different wavelength. The emission spectrum provides information for both qualitative and quantitative analysis.

When certain chemical substances are excited electronically by absorption of UV or visible radiation, they emit light at a longer wave length. This phenomenon is called luminescence.

Depending on the life span of the excited species, two different processes could be distinguished. these are fluorescence and phosphorescence.

Fluorescence: When the luminescence stops within 10-8^ to 10-4^ sec after the source of excitation is removed, it is called fluorescence.

Phosphorescence: When the luminescence continues for a slightly longer period of time (»10-4^ to 10 sec) after the source of excitation is removed, it is called phosphorescence.

Theory / Principle of fluorescence

[Excited singlet state (spins paired, no net magnetic field, avg. lifetime: 10 -8-10 -4^ sec);

Excited triplet state (spins unpaired, net magnetic field, avg. lifetime: 10 -4-10 sec)]

Molecules in the ground state

At room temp molecules reside in ground state. The ground electronic state is usually a singlet state in which all of the electrons are paired and in each pair the two electrons spin about their axis in opposite direction. The ground state can be subdivided into many states termed as vibration energy level.

Excitation and distribution of molecules in excited state

Due to absorption of UV and visible light, molecules are transmitted in the excited electronic state. Excited electronic state is also subdivided into various vibrational energy level and excited molecules will be distributed in various vibrational energy levels of excited state. Excited state is also known as excited singlet state.

Fluorescence

From the lowest vibrational energy level of the excited state, electrons can return to the ground state by photo emission and this process is known as fluorescence. Because of the vibrational relaxation, the radiation emitted as fluorescence is of lower energy & therefore, of longer wavelength than that originally absorbed.

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Internal conversion

An excited molecule may also loss energy by other processes. For example, it may undergo a radiation less loss of energy sufficient to drop to the ground state. The process is known as internal conversion. This is a competitive process of fluorescence.

Intersystem crossing

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With some compounds, another process known as intersystem crossing can also occur. Here electrons in the lowest vibrational energy level of the excited state drop down to a triplet state. The triplet state lies at an energy level intermediate between ground and excited state, and is characterized by impairing of electrons. Thus, in contrast to the singlet state, there is a spin reversal involving one electron of a pair and the two electrons spin about their axes in the same direction. Once intersystem crossing has occurred, electrons quickly drop to the lowest vibrational energy level of the triplet state by vibrational relaxation.

Phosphorescence

From the triplet state electrons can drop to the ground state by emission of radiation, this type of luminescence is termed as phosphorescence. Electrons in the triplet state can also undergo radiation less internal conversion to return to the ground state.

Fluorescence vs Phosphorescence

Fluorescence Phosphorescence

Definition Definition

It ceases as soon as the source of excitation radiation has been removed

Phosphorescence can continue after the source of excitation radiation has been removed

Energy of emitted radiation is relatively higher

Energy of emitted radiation is relatively lower

It is used as analytical tool. It is not used as analytical tool

Fluorometry VS spectrophotometry

Factors Fluorometry Spectrophotometry

Sensitivity Fluorometry is more sensitive as an analytical tool than is spectrophotometry.

Spectrophotometry is less sensitive than Fluorometry. Specificity (^) It is more specific in identifyingand analyzing a

compound than spectrophotometry.

It is less specific compared to fluorometry. Temperatur e

Intensity of fluorescence may decrease with the increase oftemperature.

Small variation of temperaturedoes not affect much. pH (^) Intensity of fluorescence is pH dependent, especially

for the solutions of weak acids and weak bases.

Small variation of pH does notaffect much.

Intensity of

incident

light

For a given concentration, the intensity of fluorescence mayincrease by increasing the intensity of the incident light.

Absorbance is independent of theintensity of the incident light.

Stability of

incident light

Intensity of incident light must be stable. Intensity of incident light is not strictly controlled.

Figure: Schematic diagram of a fluorometer

Radiation sources : It must be very intense and stable. Commonly used lamps

are mercury discharge lamp, xenon lamp etc. Excitation filter: It isolates a band of radiation to excite the sample molecules. Aglass filter is usually used. Sample holder: Glass cells are adequate for most fluorescence analysis. Quartz

cells are used below 320 nm. Emission filter: It selects a band of fluorescence emitted by the excited molecules fordetection. It is placed at the right angle to the beam of excitation radiation. Detector: It detects the intensity of fluorescence. A photomultiplier tube is used as

adetector. Recorder: The output of a detector is connected to a recorder and it records thedetector response.

Fluorometer / Spectrofluorometer

Relation between concentration of fluorescing species and

fluorescence intensity:

The intensity of fluorescence is directly proportional to the amount of light absorbed by the sample solution. F α (Io-I)…………..(i) Io=intensity of incident light I= intensity of transmitted light. Again, intensity of fluorescence is directly proportional to quantum efficiency of fluorescence (φ) F α φ……….(ii) From equation (i) and (ii) we can write F α φ (Io-I) Or F =K φ (Io-I)……(iii) From Beer’s law I=Io e -Ebc E=Molar absorptivity B=path length of irradiation C=conc. in moles per litre

Now from equation (iii) we can write F =K φ (I0-Io e – Ebc) = K φ Io (1- e – Ebc)…….(iv) At low concentration, the intensity of fluorescence is directly proportional to the conc. of the sample solution. At high conc. e – Ebc^ is about to zero, so at high conc. the intensity of fluorescence is independent of conc. Of the sample solution.

Relation between fluorescence intensity and conc.

Presence of non fluorescent impurities (Inner-filter effect)

The presence of non fluorescent solute can affect fluorescence intensity by so called inner-filter effect. In the non-fluorescent component absorb either excitation nor radiation, a reduction of in measured intensity of fluorescence will result. This effect is known as inner-filter effect.

Non fluorescent solute absorption of emission/excitation radiation

Decrease F1 intensity

Fig: inner-filter effect

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Chemical quenching

Quenching may be defined as a process in which the chemicals reduce the observed

fluorescence by the formation of complex with fluorescent molecule either in ground state

or excited state. And the chemical are called Quencher.

Chemical quenching is of two tyoes:

▸ Collision quenching

▸ Static quenching