CLINICAL CHEMISTRY

1. FLUOROMETRY
BASIC CONCEPTS,
INSTRUMENTATION AND
APPLICATIONS
By
Dr. Basil, B – MBBS (Nigeria),
Department of Chemical Pathology/Metabolic Medicine,
Benue State University Teaching Hospital, Makurdi.
September 2014.
1

2. INTRODUCTION:
• Luminescence is the emission of light by a substance. It
occurs when an electron returns to the electronic ground
state from an excited state and loses its excess energy as a
photon.
FLUORESCENCE: occurs when a molecule absorbs light at one
wavelength and reemits light at a longer wavelength
• An atom or molecule that fluoresces is termed a
Fluorophore
• Fluorometry is defined as the measurement of the emitted
fluorescence light
• Fluorometric analysis is a very sensitive and widely used
method of quantitative analysis in the chemical and
biological sciences
• Fluorescence starts immediately after the absorption of
light and stops as soon as the incident light is cut off 2

3. PHOSPHOFLUORESCENCE: This occurs when light
radiation is incident on certain substances and they emit
light continuously even after the incident light is cut off.
• Substances showing phosphorescence are
phosphorescent substances.
Fluorescence and Phosphoflourescence:
• A molecular electronic state in which all of the
electrons are paired are called singlet state.
• In a singlet state molecules are diamagnetic.
• Most of the molecules in their ground state are
paired.
• When such a molecule absorbs uv/visible radiation,
one or more of the paired electron raised to an
excited singlet state /excited triplet state. 3

4. • The electron spins in the excited state achieved by
absorption of radiation may either be parallel or
antiparallel. Accordingly, this may be a triplet
(parallel) or a singlet (antiparallel) state.
Singlet and Triplet States:
4

5. BASIC CONCEPTS: Jablonski Diagram
5

6. • Each molecule contains a series of closely spaced
energy levels
• Absorption of a quantum of light energy by a molecule
causes the transition of an electron from the singlet
ground state to one of the number of possible
vibrational levels of its first singlet state
• Once the molecule is in an excited state, it returns to
its original state in several ways. These are:
– Radiation-less vibrational equilibration
– The fluorescence process from the excited singlet state
– Quenching of the excited singlet state
– Radiation-less crossover to a triplet state
– Quenching of the first triplet state, and
– The phosphorescence process of light emission from the
triplet state. 6

7. • The vibrational equilibrium before fluorescence
results in some loss of the excitation energy,
• thus, the emitted fluorescence is of less energy
and longer wavelength than the excitation light
• The difference btw the max wavelength of the
excitation light and the max wavelength of the
emitted fluorescence lights is a constant – stokes
shift
• This constant is a measure of energy lost during
the lifetime of the excited state (RVD) before
returning to the ground singlet level (fluorescence
emission).
7

8. Time Relationship of Fluorescence Emission:
• There is a considerable time delay btw the (1)
absorption of light energy, (2) return to the lowest
excited state, (3) emission of fluorescence light.
8

9. • The time required for the emitted light to reach 1/e of its
initial intensity , where e is the Naperian base 2.718, is called
the average lifetime of the excited state of the molecule, or
the fluorescence decay time
• The advantage of time-resolved fluorometer (time delay btw
absorption of quanta of light and fluorescence) is the
elimination of background light scattering as a result of
Rayleigh and Raman signals and Short-lived fluorescence
background
• Time-resolved fluorometry depending on how the
fluorescence emission response is measured is categorized
as:
– Pulse flurometry: sample illuminated with as intense brief pulse
of light and the intensity of emission measured as a function of
time with a fast detector
– Phase fluorometry: continuous-wave laser illuminates the
sample, and the emission monitored for impulse and freq
response.
9

10. Relationship of concentration and fluorescence
intensity:
• Fluorescence intensity is directly proportional to the
concentration of the fluorophore and the excitation
intensity
F = ФIo abc
where F = relative intensity
Ф = fluorescence efficiency (i.e., the
ration btw quanta of light emitted
and quanta of light absorbed)
Io = initial excitation intensity
a = molar absorptivity
b = volume element defined by geometry of
the excitation and emission slits
c = the concentration in mol/L 10

11. • This relationship holds only for dilute solutions, where
absorbance is less than 2% of the exciting radiation.
Higher than 2%, the fluorescence intensity becomes
nonlinear – inner filter effect.
• The magnitude of fluorescence intensity of a
fluorophore is determined by:
– Its concentration
– The path length
– The intensity of the light source
• Fluorescence measurements are 100 – 1000 times
more sensitive than absorbance measurement due to:
– More intense light source
– Digital filtering techniques
– Sensitive emission photometers
11

12. • Fluorescence measurements are expressed in “relative”
intensity units because the intensity measured is not an
absolute quantity and its magnitude is defined by the
(instrument-related varriables):
– Instrument slit width
– Detector sensitivity
– Monochromator efficiency
– Excitation intensity
Fluorescence Polarization:
• This is defined by:
P = (Iv – Ih)/(Iv + Ih)
where Iv = intensity of emitted fluorescence light in vertical
plane
Ih = intensity of emitted fluorescence light in horizontal
plane
• Fluorescent polarization is measured by placing a
mechanically or electrically driven polarizer btw the sample
cuvet and detector
12

13. 13

14. • In normal instrumentation mode, the sample is excited
with polarized light to obtain maximum sensitivity.
• The polarizer analyser is placed to measure the
intensity of the emitted fluorescence both in vertical
and horizontal planes before calculating for P.
• Fluorescence polarization is used to quantitate
analytes by use of the change in fluorescence
depolarization following immunological reactions
• This is done by adding a known quantity of
fluorescent-labelled analyte molecule to a reaction
solution containing an antibody specific to the analyte.
• The labeled analyte binds to the antibody resulting in a
change in its rotational relaxation time and
fluorescence polarization.
14

15. QUENCHING:
• Decrease in fluorescence intensity due to specific
effects of constituents of the solution e.g.,
concentration, ph, pressure of chemical
substances, temperature, viscosity, etc.
• Types of quenching include:
– Self quenching
– Chemical quenching
– Static quenching
– Collision quenching
Collisional Quenching:
• It reduces fluorescence by collision. where no. of
collisions increased hence quenching takes place.15

16. Self Quenching/ Concentration Quenching:
16

17. Chemical Quenching:
• Here decrease in fluorescence intensity due to the
factors like change in ph, presence of oxygen, halides
& heavy metals.
• pH- aniline at pH 5-13 gives fluorescence but at pH <5
&>13 it does not exhibit fluorescence.
• halides like chloride, bromide, iodide & electron
withdrawing groups like NO2, COOH etc. leads to
quenching.
• Heavy metals leads to quenching, because of
collisions of triplet ground state.
Static Quenching:
• This occurs due to complex formation e.g., caffeine
reduces the fluorescence of riboflavin by complex
formation.
17

18. PHOTODECOMPOSITION:
• Excitation of a weakly fluorescing or dilute solutions
with intense light sources will cause photochemical
decomposition of the analyte (Photobleaching). This
is minimized by:
– Use of the longest feasible wavelength for excitation that
does not introduce light-scattering effects.
– Measure the fluorescence immediately after excitation to
decrease the duration of excitation
– Protect unstable solutions from ambient light by storing
them in dark bottles
– Remove dissolved oxygen from the solution
• Decomposition introduces non-linear response curves
and loss of the majority of the sample fluorescence.18

19. Factors affecting Fluorescence and
Phosphorescence
Temperature/Viscosity:
• 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.
• A rise in temperature is almost always accompanied by
a decrease in fluorescence.
• Increase in viscosity increases fluorescent intensity
• The increase in the number of collisions between
molecules in turn increases the probability for
deactivation by internal conversion and vibrational
relaxation (Collisional Quenching).
• Temperature of the reaction must be regulated to
within +/- 0.1’C 19

20. 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.
• In the molecules containing acidic or basic functional groups,
the changes in pH of the medium change the degree of
ionisation of the 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.
20

21. • Therefore, pH control is essential while working with
such molecules and suitable buffers should be
employed for the purpose.
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.
• Presence of dissolved oxygen influences phospho-
rescence too and causes a large decrease in the
phosphorescence intensity. 21

22. • It is due to the fact that oxygen 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.
Solvent:
• The changes in the “polarity” or hydrogen bonding
ability of the solvent may also significantly affect the
fluorescent behaviour of the analyte.
• The difference in the effect of solvent on the
fluorescence is attributed to the difference in their
ability to stabilise the ground and excited states of the
fluorescent molecule. 22

23. • Besides solvent polarity, solvent viscosity and solvents with
heavy atoms also affect fluorescence and phosphorescence.
• 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.
• Some solvents e.g Ethanol, also cause appreciable
fluorescence.
• Other sample matrix, e.g protiens and bilirubin are more
serious contributors to unwanted fluorescence
Adsorption:
• Extreme sensitiveness of the method requires very dilute
solution. Adsorption of the fluorescent substances on the
container wall create serious problems. Hence strong
solutions must be diluted.
• Certain quartz glass and Plastic materials that contain
ultraviolet adsorbers will fluoresce 23

24. Concentration Effects:
Concentration is proportional to the emitted light energy
absorbed . At the maximum concentration, fluorescence
peaks and may decrease thereafter.
• Concentration quenching
• Inner Filter Effect: refers to the deviation from a linear
relationship btw concentration and fluorescence as the
absorbance of the solution increases above 2% of the exciting
light
– Thus, as the concentration increases, the absorbance of the
excitation intensity increases, and the loss of the light as it travels
through the cuvet increases.
Light effects:
• Type of light: Monochromatic light is essential for the excitation
of fluorescence because the intensity will vary with wavelength.24

25. • Length of time of exposure: The number of molecules
excited increase with the time of exposure hence
decrease in fluorescence due to Photodecomposition
• Intensity of Incident light: Increase in intensity of light
incident on sample increases fluorescence intensity.
The intensity of light depends upon:
– Light emitted from the lamp.
– Excitation monochromaters.
– Excitation slit width
• Light scattering:
– Rayleigh scattering: Diffusion of radiation in the course
of its passage through a medium containing particles
the size of which is small compared with the
wavelength of the radiation
• occurs with no change of wavelength
– Raman scattering occurs with lengthening of
wavelength.
• It is a property of the solvent and difficult to eliminate at low
fluorophore concentration 25

26. Nature of Substituent:
• Electron donating group enhances fluorescence –
.g.NH2,OH etc.
• Electron withdrawing groups decrease or destroy
fluorescence. e.g.COOH,NO2, N=N etc.
• High atomic no: atom introduced into the electron
system decreases fluorescence.
Path length:
• The effective path length depends on both the
excitation and emission slit width.
• Use of microcuvette does not reduce the
fluorescence.
• Use of microcell may reduce interferences and
increases the measured fluorescence
26

27. INSTRUMENTATION:
THE FLUOROMETER
Single-beam and Double-beam Instrument Designs
27

28. Basic Components:
• Basic components of fluorometers and Spectrofluorometers
include:
– an excitation source, an excitation monochromator, a cuvet, an
emission monochromator, a detector.
• The optical geometry for fluorescence measurement is very
important:
– most commercial fluorometers use the right angle detector
approach because it minimizes the background signal that limits
analytical detection.
– The end-on approach allows the adaptation of a fluorescence
detector to existing 180 degrees absorption instrument.
– The front surface approach has a comparable limit of detection
to right angle detectors and it minimizes the inner filter effect,
but is more susceptible to background light scatter.
• To accommodate these different geometries, the sample
cell is oriented at different angles in relation to the
excitation source and the detector.
28

29. Basic Components of Fluorometers:
29

30. Geometrical arrangements of Fluorometres:
30

31. • If the emission slit is located near the front edge of the
sample cell, the inner filter effect is minimized.
• If the emission slit width is increased, the detector will
be more sensitive, but specificity may decrease.
31

32. Light Source:
• Mecury Arc Lamp:
– Produce intense line spectrum above 350nm.
– High pressure lamps give lines at 366,405, 436,
546,577,691,734nm.
– Low pressure lamps give additional radiation at 254nm.
• Xenon Arc Lamp:
– Intense radiation by passage of current through an
atmosphere of xenon.
– Spectrum is continuous over the range between over 250-
600nm,peak intensity about 470nm
• Tungsten Lamp:
– Intensity of the lamp is low.
– If excitation is done in the visible region this lamp is used.
– It does not offer UV radiation
• Turnable Dye Lasers:
– Pulsed nitrogen laser as the primary source
– Radiation in the range between 360 and 650 nm is
produced. 32

33. • Excitation monochromators:- isolates only the
radiation which is absorbed by the molecule.
• Emission monochromaters:- isolates only the radiation
emitted by the molecule.
• Primary filter:- absorbs visible light & transmits UV
light.
• Secondary filter:- absorbs UV radiations & transmits
visible light.
Cuvets:
• Cylindrical or rectangular cells fabricated of silica or
glass used.
• Path length is usually 10mm or 1cm.
• All the surfaces of the sample holder are polished in
fluorimetry.
Monochromators and Filters:
33

34. Detectors:
• These include:
– Photovoltaic Cell
– Phototube
– Photomultiplier tube: - best and accurate
• Photomultiplier Tube:
– Multiplication of photo electrons by secondary
emission of radiation.
– A photo cathode and series of dynodes are used.
– Each cathode is maintained at 75-100v higher than the
preceding one.
– Over all amplification of 106 is obtained.
34

35. Single-Beam Fluorometers:
• Tungsten lamp as source of light.
• The primary filter absorbs visible radiation and transmits
uv radiation.
• Emitted radiation measured at 90o by secondary filter.
• Secondary filter absorbs uv radiation and transmits visible
radiation.
Advantages:
• Simple in construction, easy to use and economical
Disadvantages:
• It is not possible to use reference solution & sample
solution at a time.
• Rapid scanning to obtain Exitation & emission spectrum of
the compound is not possible.
35

36. Double-Beam Fluorometer:
• Two incident beams of light source pass through
primary filters separately and fall on either sample
or reference solution
• The emitted radiation from sample or reference
pass separately through secondary filter.
Advantages:
• Sample and Reference solution can be analysed
simultaneously
Disadvantage:
• Rapid scanning is not possible due to use of filters.
36

37. APPLICATIONS:
• Determination of Vitamin B1 and B2
• Liquid Chromatography:
– It is an important method of determining compounds as
they appear at the end of chromatogram or capillary
electrophoresis column
• Organic Analysis:
– Qualitative and quantitative analysis of organic aromatic
compounds present in cigarette smoke, air pollutants,
automobile exhausts etc.
• Fluorescent indicators:
– Mainly used in Acid-Base titration e.g:
• Eosin: colourless – green
• Fluorescein: colourless – green
• Quinine sulphate: blue – violet
• Acridine: green – violet. 37

38. • Fluorometric reagent:
– Aromatic structure with two or more donor functional
groups
• Pharmaceutical Analysis:
38

39. • Other Applications include:
– Determination of inorganic substances: like Aluminum,
boron, cadmuim with 2-(2OH-phenyl)benzoxazole in
the presence of tartarate, ruthenuim
– Determination of Uranuim salts in Nuclear research
39

40. REFERENCES:
• Teitz Textbook of Clinical Chemistry and Molecular
diagnosis (5th Edition)
• Dr.B.K.Sharma, Instrumental methods of chemical
analysis.
• Gurdeep R Chatwal, Instrumental methods of
chemical analysis
• http://en.wikipedia.org/wiki/Fluorescence
• http://images.google.co.in/imghp?oe=UTF-
8&hl=en&tab=wi&q=fluorescence
• http://www.bertholdtech.com/ww/en
pub/bioanalytik/biomethods/fluor.cfm 40

41. THANK YOU
FOR
LISTENING
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