MAT

1. SPECTROFLOURIMETRY
Modern Pharmaceutical Analytical Techniques
1

2. CONTENTS
 Introduction
 Definition
 Theory
 Factors affecting fluorescence
 Instrumentation
 Application
 Conclusion
 Reference
2

3. INTRODUCTION
 Absorption of UV-VIS radiation causes transition of
electrons from ground state to excited state.
 As excited state is not stable so, excess energy is released
by
 Collisional deactivation
 photoluminescence
 This study or measurement of this emitted radiation is the
principle of Flourimetry.
 Phosphorescence is also related phenomena, which is
study of emitted radiation when electron undergoes
transition from triplet state to singlet ground state .
3

4. DEFINITION
 Singlet ground state: a state in which all the electrons in a
molecule are paired (↑↓)
 Doublet state: a state in which an unpaired electron is
present e.g., free radical ↑ or ↓
 Triplet state: a state in which unpaired electrons of same
spin present ↑ ↑ (unpaired and same spin)
 Singlet excited state: a state in which electrons are
unpaired but of opposite spin like ↑ ↓ (unpaired and
opposite )
4

5.  Collisional deactivation: in which entire energy is lost due
to collisional deactivation and no radiation is emitted.
 Fluorescence: a part of energy is lost due to vibrational
transitions and remaining energy is emitted as UV-VIS
radiation of longer wavelength.
 Fluorescence is the phenomena of emission of radiation
when the is transition from single excited state to singlet
ground state.
 The wavelength of absorbed radiation is called excitation
wavelength.
 The wavelength of emitted radiation is called emission
wavelength.
5

6.  Phosphorescence: at favourable conditions like low temp.
and absence of oxygen there is transition from excited
singlet state to triplet state which is called as inter
system crossing. The emission of radiation when electrons
undergoes transition from triplet state to singlet ground
state is called as phosphorescence .
6

7. Fig:- Phenomena of fluorescence and phosphorescence.
7

8. THEORY
 Both fluorescence and phosphorescence are types of
photoluminescence (luminescence)
 Luminescence is described by using a molecular-energy
interpretation.
 Fluorescence of organic molecules means emission of radiant
energy during a transition from the lowest excited singlet state
𝑆1 to the singlet ground state 𝑆0 .
 Phosphorescence of organic molecules means emission of
radiant energy during a transition from the lowest excited
triplet state 𝑇1 to the singlet ground state 𝑆0 .
8

9.  In phosphorescence, an intersystem crossing can take
place readily from 𝑆1 to one of the vibrational levels of 𝑇1
state that has very nearly the same energy level (process
III). This is followed by non radiative decay (process IV) to
the 𝑇1 level.
9

10. Fig; Partial energy diagram for a photo luminescent system
10

11.  Intersystem crossing process involves a change in the spin of
the excited electron and thus a change in spin multiplicity.
 The triplet state 𝑇1 is metastable, and molecules populating
it have excess energy. This energy can be lost by
 Phosphorescence (process V)
 Oxygen quenching:
 By collision
11

12. Differences between fluorescence and
phosphorescence
 Phosphorescence may sometimes persist for many seconds after
the excitation source is removed.
 Fluorescence emission is always at shorter wavelength than that
of phosphorescence.
 Fluorescence is usually observed at room temperature in liquid
solution, while phosphorescence is observed in rigid medium at
very low temperature.
 Fluorescence life time is usually in the range 10-7-10-9 sec, while
phosphorescence lifetime is usually in the range 10-4 -10 sec.
12

13.  Half life time: It is the time required for half of the
molecules to emit photons and thus return to the ground
states.
 Effect of molecular structure on luminescence properties
13

14. Fluorescence may be expected
generally in;
 Aromatic molecules that contain conjugated double
bonds
 Polycyclic aromatic compounds (with great number of π
electrons)
 Substituents strongly affect on the fluorescence;
substituents such as NH2, NHCH3, N(CH3)2, OH and OCH3
groups enhance the fluorescence, while electron with
drawing group such as NO2 Cl-, Br-, I- and COOH groups
decrease the fluorescence
 Formation of metal chelates promotes the fluorescence.
14

15. Phosphorescence may be expected
generally in
 Aromatic hydrocarbons
 Introduction of substituents such as NH2, SH, OH to
aromatic hydrocarbon enhance the phosphorescence
and also aromatic nitro compounds
 Majority of aromatic aldehydes and ketones show
phosphorescence.
15

16. Fluorescence Spectra
Instruments that measure the intensity of fluorescence are called
fluorimeter. Those that measure the fluorescence intensity at
variable wavelengths of excitation and emission, and are able to
produce fluorescence spectra are called spectrofluorimeters
In the recording the fluorescence spectra, the limitations of light
sources and measuring devices assume real significance. These
limitations are:
 Variation of the intensity of available energy with λ.
 Variation in the response of the detector to light of
different wavelengths
16

17. .
Excitation Spectra:
Before a compound can fluoresce, energy must be observed, and
with an ideal light source, of constant intensity at different
wavelengths, the most intense fluorescence is produced by
radiation corresponding in wavelength to that of the absorption
peak of the substance. Therefore, if the intensity of the
fluorescence is plotted as a function of the wavelength of the
radiation used to excite the fluorescence, an activation or
excitation spectrum will, result. This will be identical to the
absorption spectrum when corrected for instrumental effect,
because the fluorescence efficiency is greatly independent of λ .
17

18. Fig; Fluorescence excitation and emission spectra for a solution of
quinine
18

19. Emission Spectra (Fluorescence)
When a monochromator source of constant light intensity is
used to irradiate a sample, the fluorescence may be
analysed in a monochromator at constant slit width to give
apparent emission spectrum.
The true spectrum is obtained by applying a correction for
change in detector sensitivity with wavelength and for
changes due to fluorescence monochromator i.e., half band
width of emergent light and light losses.
Fluorescence emission spectra arise because of transition
from the first excited state and their shapes are therefore
independent of the light used to excite fluorescence.
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20. FACTORS AFFECTING FLUORESCENCE
INTENSITY;
 Concentration
 Quantam yield
 Intensity of incident light I0
 Pathlength (b)
 Adsorption
 Oxygen
 pH
 Temperature and viscosity
 Scatter
20

21. Concentration:
The fluorescence intensity of a substance is proportional to
concentration only when the absorbance in a 1 cm cell is less than
0.02.
If the concentration of the fluorescent substance is so great that all
incident radiation is absorbed, the equation will be:
F = I0
ϕ
That is the fluorescence is independent of concentration, and
proportional to the intensity of incident radiation only, a property that
may be utilized to determine the approximate emission characteristics
of a light source
21

22. Diagrammatic representation of the variation of fluorescence intensity
with concentration.
Region (a): Proportional relationship
Region (b): Negative deviation from linearity.
Region (c): Fluorescence independent of concentration
Region (d): Reabsorption of fluorescence 22

23. Quantum yield of fluorescence (ϕ)
This is the ratio:
ϕ =
𝑛𝑜.𝑜𝑓 𝑝ℎ𝑜𝑡𝑜𝑛𝑠 𝑒𝑚𝑖𝑡𝑡𝑒𝑑
𝑛𝑜.𝑜𝑓 𝑝ℎ𝑜𝑡𝑜𝑛𝑠 𝑎𝑏𝑠𝑜𝑟𝑏𝑒𝑑
Since some absorbed energy is lost by radiation less pathways,
the quantum efficiency is less than 1.
Highly fluorescent substances take ϕ value near 1, which
shows that most of the absorbed energy is re-emitted as
fluorescence.
For e.g.; fluorescein in 0.1 M NaOH and quinine in 0.05 M
H2SO4 have, ϕ values of 0.85 and 0.54 respectively at 23°C.
Non-fluorescent substances have ϕ = 0.
23

24. Intensity of incident light 𝐈 𝟎
:
An increase in the intensity of light incident on the sample
produces a proportional increase in the fluorescence
intensity. The intensity of incident light depends on the
intensity of light emitted from the lamp.
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25. Pathlength (b)
The effective pathlength viewed by the detector depends on
both the excitation and emission slit widths.
25

26. Adsorption:
The extreme sensitivity of the method requires very dilute
solution, 10-100 times, weaker than those employed in absorption
spectrophotometry.
Adsorption of the fluorescent substance on the container walls
may therefore presents serious problems and strong stock solutions
must be kept and diluted as required. Quinine is a typical example
of a substance which is adsorbed onto cell walls.
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27. Oxygen
The presence of oxygen may interfere in two ways:
 By direct oxidation of the fluorescent substance to non-
fluorescent products.
 By quenching of fluorescence.
27

28. pH
 It is to be expected that alteration of the pH of a solution will
have a significant effect on fluorescence if the absorption
spectrum of the solute is changed.
28

29. Temperature and viscosity
Variation in temperature and viscosity will cause variations
in the frequency of collision between molecules. Thus, an
increase in the temperature or the decrease in the viscosity
is likely to decrease the fluorescence by deactivation of the
excited molecules by collision.
29

30. Quenchers
Quenching is the reduction of the fluorescence intensity by
the presence of substances in the sample other than the
fluorescent analyte.
 Static Quenchers:
Form a chemical complex with the fluorescent substance
and alter its fluorescence characteristics. Certain xanthine
derivatives e.g. caffeine, reduce the fluorescence of
riboflavin by static quenching.
30

31. Scatter
The excitation and emission monochromators are at the
same wavelengths, scattered light of the same wavelength
as the incident light will be detected by the photomultiplier
arising from colloidal particles in the sample (Tyndall
scatter) and from the molecules (Rayleigh scatter)
31

32. .
Raman Scatter:
Arises from the conversion of some of the incident radiation
into vibrational and rotational energy by the solvent
molecules. The resultant scattered light is of lower energy
and, consequently of longer wavelength.
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33. INSTRUMENTATION
When both the excitation and emission spectra are to be
recorded, two monochromator are essential, one for the light source
(excitation monochromator) and one for the fluorescence (emission
monochromator). The light source must provided a high level of UV
and Visible radiation and a compact high pressure Zenon arc lamp is
used.
The production of ozone by the photochemical conversion of
atmospheric oxygen in the lamp compartment presents a toxic
hazard unless the ozone is thermally decomposed or removed by
adsorption onto charcoal. As many experiments will almost certainly
entail the measurement of very weak fluorescence. The detector
must be a highly sensitive photomultiplier tube of low dark current.
If the main interest lies in the fluorescence emission spectra, one
monochromator may be dispensed with a suitable light source33

34. and filter used instead. The rather poor luminosity
associated with the monochromator even with a xenon arc
lamp is replaced by the much more intense light from a
source such as a mercury vapour lamp, from which a suitable
activation beam is isolated by means of the filter. This
arrangement partially overcome, one of the difficulties
inherent in spectrofluorimetry, i.e., that so much of the
available light is lost.
34

35. Fig; simple fluorometer 35

36. ADVANTAGES OF SPECTROFLUORIMETRY
 High sensitivity
 Selectivity
 Quantitative Aspects
36

37. Quantitative Aspects:
Many of the quantitative aspects of spectrofluorimetry may be
understood by reference to the fundamental equation for the
intensity of the fluorescence emitted. This equation may be
derived from that of the
Beer-Lambert law:
A = Log 𝑰 𝟎/𝑰 𝒕 = abC
Or 𝑰 𝟎/𝑰 𝒕 = 𝟏𝟎 𝒂𝒃𝒄
∴ 𝑰 𝒕= 𝑰 𝟎 x 𝟏𝟎−𝒂𝒃𝒄
37

38. but fluorescence (F) = (𝑰 𝟎- 𝑰 𝒕)ϕ
where ϕ is the quantum yield of the fluorescence
At very low absorbance (< 0.02), the equation will be
F= 2.3 𝑰 𝟎 abC ϕ
For a fixed set of instrumental (𝐼0 and b) and sample (a
andϕ) parameters, the fluorescence is proportional to the
concentration.
F= K C where K = 2.3 𝑰 𝟎 a b ϕ
38

39. APPLICATIONS OF SPECTROFLUORIMETRY
 Compounds which are fluorescent are readily determined with
simple instruments as the solution for examination is normally
obtained by dissolution of the sample in a suitable solvent
(table 1)
39

40.  Single substances which are in themselves, non-
fluorescent may be determined as a result of chemical
change.
e.g.;
Determination of primary amines, amino acids, peptides
etc. through:
40

41. Reaction with fluorescamine reagent
41

42.  Determination of primary and secondary aliphatic amines
through:
a- reaction with 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole (
NBD-CI ) give yellow fluorescence
42

43. b- reaction with l-dimethylaminonaphthalene-5-sulphonyl
chloride (Dansyl, chloride)
43

44. Thiamine HCI in pharmaceutical preparations such as tablets
and elixirs and in food stuffs such as flour is relatively easily
determined by oxidation to highly fluorescent thiochrome.
The product is soluble in 2-methyl-propan-1-ol and hence is
easily extracted from the reaction mixture for
measurements
44

45. For mixture of two components, it may be possible to select
the exciting radiation of appropriate wavelengths, such that
only one compound fluoresces at any time. Even if there is
not possible, measurements of the fluorescence at two
wavelengths may be sufficient to determine the composition
of the mixture.
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46. CONCLUSION
 Fluorescence is most sensitive analytical techniques.
 Detection studies will increase the development of
fluorescence field.
 Flurometric method are much useful in qualitative
analysis as compared to quantitative analysis.
46

47. REFERENCES
1. Douglas a, Skoog , Holler, PRINCIPLE OF INSTRUMENTAL
ANALYSIS, 5th edition , Saunders college, west Washington
square, Philidhepia
2. Dr Ravishankar , A TEXTBOOK OF PHARAMACEUTICAL
ANALYSIS, 3rd edition, Rx pub., 57, west car street,
Tiruneveli-627006
3. Gr Chatwal , S.K. Anand, INSTRUMENTAL METHOD OF
CHEMICAL ANALYSIS, Himalaya pub. house pvt ltd, Ramdoot',
Dr. Bhaleraomarg, girgaon, Mumbai - 400 004, Maharashtra,
India
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48. THANK YOU
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