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Fluorescence Spectrometry

The document discusses fluorescence spectrometry, detailing its principles, instrumentation, factors affecting fluorescence intensity, and various applications. It explains the processes of fluorescence and phosphorescence, the roles of different components in spectrometers, and the significance of factors like molecule nature and concentration. Applications range from quantitative analysis in biological and chemical sciences to detecting environmental pollutants and analyzing drugs in medicine and pharmacy.

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zainzulfiqar0410
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12 views7 pages

Fluorescence Spectrometry

The document discusses fluorescence spectrometry, detailing its principles, instrumentation, factors affecting fluorescence intensity, and various applications. It explains the processes of fluorescence and phosphorescence, the roles of different components in spectrometers, and the significance of factors like molecule nature and concentration. Applications range from quantitative analysis in biological and chemical sciences to detecting environmental pollutants and analyzing drugs in medicine and pharmacy.

Uploaded by

zainzulfiqar0410
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Fluorescence Spectrometry/Spectrofluorometry, it’s principle,

instrumentation, factors and applications

Asad Mushtaq-075, Ali Azhar- , Wallail Ahmed- , Zain Zulfiqar-


116, Zain Abbas- ,

Introduction

Fluorescence is a phenomena of light emission in which the energy transit


from higher state to lower state, within the molecule/fluorophore, that is
measure by the detection of emitted radiation as absorbed radiation.
Measurement of emitted fluorescent light is known as fluorometry. Light
absorb by fluorophore at one wavelength and light reemits at lomger
wavelength.

Principle

When UV light fall on sample, it will absorb UV light (short wavelength 200-
400nm) but when excited, atom gain stability and move to ground state it
will emits light (longer wavelength 400-800nm). This emission leads to
fluorescence/phosphorence. Electron move around its orbital either in
clockwise or anti-clockwise direction. When two electrons are in same shell,
according to huntz rule one electron move in clockwise direction and other
will move in anti-clockwise direction.

Singlet state

When UV light absorb, two electrons of same shell moving in different


direction, one of them is excited to higher orbital, E.g electron which was
moving in anti-clockwise direction if move to higher orbital, it remain
moving in anticlockwise direction. When it move back to ground state it
emit visible region light.

Singlet state=+1/2 , -1/2

Net spin=0
Spin multiplication= 2n+1 2(0)+1=1

Triplet state

Two electrons paired in an orbital, when UV light is absorb, one electron


will move to higher orbital and change its spin E.g if electron moving within
an orbital in anti-clockwise excite to higher orbital it will change its spin to
clockwise direction. When it move back to ground state it will change its
spin as earlier (anti-clockwise).

Due to environmental or other factor spin change change in excited state.

Triplet = +1/2 , -1/2 (in ground state)

In excited state = +1/2 , +1/2

Net size = 1

Spin multiplication = 2n+1 = 2(1)+1 = 3

Triplet is more stable than singlet state, triplet state has more unpaired
electrons then singlet state and triplet state electrons pointed in same
direction while singlet state electrons move in opposite direction. Triplet
state also lower in energy as siglet state.
Phosphorescence

In triplet, highly stable state. Both electrons in ground and excited are in
same spin take time to move in ground state. Excited electrons change its
spin as earlier when move in ground state.

Fluorescence Instruments

1. Excitation source
2. Excitation monochromator
3. Cuvet
4. Emission monochromator
5. Detector
Excitation source

Initial excitation intensity, concentration and size of sample cell is


directly proportional to intensity of florescence emission. An intense
lamp has ability to emitting radiant energy.

E.g Xenon lamp, Mercury arc lamp, Quartz halogen and Laser.

Excitation and emission monochromator

Two monochromators are used

1. Turning exciting beam wavelength.


2. Analyse the emission fluorescence emission.

Light always have low energy than exciting due to emission, wavelength
is set at lower in exciting monochromator as emitting monochromator.
Interference filters, colored glass filters, gratings and prisms are
combined with sharp cutog glass filters to form single package filter that
remove undesired transmission, provide narrow bandwidth, higher peak
transmission wavelength and increased lope band.

Cuvet
Cuvet is same as with spectrophotometers, cuvet and excitation beam
place with spectroflorometers to the photodetector is establish optical
geometry for measurement of florescence. Most spectroflorometrs are
use in practice due to its ability to minimize the background signal that
limits sensitivity of analysis.
I. Front surface of cuvet provide great
linearity due to minimizing of inner filter effect. Its widely used in
heterogeneous solid phase florescence. C

ExM

EmM

Photodetector

There are two types of detector are used in fluorescence spectrometer

1. Photomultiplier tube (PMT)


2. Chargecoupled detector (CCD)

Photomultiplier tube (PMT)


Common used detector in spectroflorometer is PMT due to having
important features like wide spectral responses, nanoseconds photon
response and sensitivity.

Charged-coupled detector (CCD)

CCD is multichannel devices with signal-to- noise ratio and dynamic range
that is superior to PMT.

CCD is used fot molecular florescence measurement for very low


concentration florescent molecules due to ability to detect light at very low
level.

Factor affecting fluorescence intensity

1. Nature of molecule
2. Nature of substituent
3. Effect of concentration
4. Absorption of light
5. pH of sample and oexygen
6. Photodecomposition
7. Temperature and viscosity of sample
8. Intensity of incident light
9. Path length
Application

1. It is quantitative analysis method widely used in biological and


chemical sciences.
2. It is used to detect pollutants present in environment such as
polycyclic aromatic hydrcarbons pyrene, benzopyrene and carbamate
insectisides.
3. Used in analytical chemistry:

Detect compound in HPLC flow

TLC plates can be visualized if colouring reagents used

Plant pigments, protiens, steroids, naphthols etc.

4. Used in Biochemistry

Analysis of biological molecules (protiene)

Direct or indirect analysis of aromatic aminoacids (phenylalanine,


tyrosine, tryptophan)

5. Medicine
6. In pharmacy

Direct or indirect analysis of drugs.

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