Molecular Spectroscopic

Methods-II

UNIT 6 APPLICATIONS OF FLUORIMETRY

AND PHOSPHORIMETRY
Structure
6.1

Introduction

6.2

Fluorescence Analysis Methods

Objectives
Direct Analysis Methods
Indirect Analysis Methods

6.3

Fluorescence Spectroscopy in Quantitative Analysis

Concentration Dependence of Fluorescence
Factors Affecting Quantitative Applications of Fluorimetry

6.4

Fluorimetry and Environmental Monitoring

Analysis of Gaseous Pollutants
Analysis of Water Pollutants

6.5

Fluorescence Spectroscopy in Medicine and Biology

Analysis of Amino Acids and Proteins
Fluorimetric Determination of Blood Glucose
Analysis of Blood Serum
Analysis of Creatinine Phosphokinase
Analysis of Calcium Ion
Bioluminescence

6.6

Fluorimetric Analysis of Inorganic Substances

Chemical Reactions Producing Fluorescence
Inorganic Substances Showing Luminescence
Fluorescence with Inorganic Reagents
Fluorescence with Organic Reagents

6.7
6.8

Fluorescence and Mineral Analysis

Phosphorimetric Methods in Chemical Analysis
Room Temperature Phosphorescence
Applications of Phosphorescence Measurements

6.9 Summary
6.10 Terminal Questions
6.11 Answers

6.1

INTRODUCTION

In the previous unit, you have studied about the fluorescence and phosphorescence
spectroscopies. You have learnt that these techniques can be categorised as the
examples of molecular emission spectroscopy. You have also learnt about the
difference between fluorescence and the phosphorescence phenomena and how
transition time makes a radical difference in the utility of these two methods.
Phosphorimetry lags behind fluorescence as an analytical tool because of lack of
suitable instruments. However, with advent of time, greater strides have been made in
the field of instrumentation and in the last two decades great progress has been made
in the field of phosphorescence spectroscopy. The newer methods have shown that
measurements at room temperature instead of at low temperature can widen the
domain of analytical phosphorimetry.
In this unit, you would learn about important applications of fluorescence and
phosphorescence spectrometry in quantitative and qualitative analysis of a variety of
inorganic as well as organic compounds. In addition, we would take up some specific
applications in the areas of environmental and biochemical analysis. You would learn

28

how we can effectively go down to the concentrations in the range of nanogram to

picogram (ng-pg) of the substances to be analysed by these methods.

Applications of
Fluorimetry and
Phosphorimetry

Objectives
After studying this unit, you will be able to:

list the applications of fluorescence and phosphorescence measurements in

chemical analysis,

describe the analysis of gaseous pollutants by chemiluminescence,

discuss the analysis of water pollutants by fluorimetric methods,

state the principle behind the analysis of biological samples using

photoluminescence methods,

give examples of the organic complexing ligands producing fluorescence with

metals,

enumerate the types of reactions producing fluorescence,

list factors responsible for fewer analytical applications of phosphorimetry, and

discuss the importance of room temperature phosphorescence

6.2

FLUORESCENCE ANALYSIS METHODS

The applications of fluorimetry and phosphorimetry to the physical and life sciences
have evolved rapidly during the past decade. The increased interest in these appears to
be due to advances in time resolution, methods of data analysis, and improved
instrumentation, etc. In addition, the advances in laser and detector technology have
also contributed towards the renewed interest in fluorescence measurements in clinical
and analytical chemistry. The key characteristic of fluorescence spectrometry is its
high sensitivity. It may achieve detection limits of several orders of magnitude lower
than most of the other techniques. Due to the low detection limits, fluorescence is
widely used for quantification of trace constituents of biological and environmental
samples. Before taking up the applications of fluorescence in details it is worthwhile
to learn about the basic fluorescence analysis methods. There are two types of
fluorescence analysis methods i.e. direct analysis methods and indirect analysis
methods as described in the following sub-sections.

6.2.1

Direct Analysis Methods

In direct methods, we measure the natural fluorescence of the analyte. A few inorganic
species fall under this category whereas the number of the organic species in this
category is quite large. In the category of inorganic species the lanthanides; cerium
and europium being the most common examples, actinides; uranium being the most
important and some other ions like thallium show reasonable intrinsic fluorescence to
be determined by the direct method. However, the sensitivities of these determinations
are not very good.

6.2.2

Indirect Analysis Methods

As is obvious from the name, in indirect analysis methods the fluorescence is not
measured directly. There are two strategies of indirect methods of analysis. In the first
strategy the analyte to be determined is suitably derivatised to a fluorescent species.
For example, 2-hydroxybenzaldehyde (salicyaldehyde) forms bonds with metals and
the resulting species is fluorescent as shown.

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Molecular Spectroscopic
Methods-II

C
O
H

O
Salicyaldehyde

M+
O

H+

Salicyaldehyde bonded with metal

The origin of fluorescence in the molecule may be attributed to the elongation of the
chromophore and also to the formation of a ring. The determination of selenium is
another example of indirect determination. In this case diaminonaphthalene is used as
a fluorescent label. In indirect methods of analysis, it is desirable that the metal ion to
be determined and the ligand, both are nonfluorescent. This facilitates the
measurement of the fluorescence of the complex. In case one of the components is
fluorescent, it must have very weak fluorescence.
In the second strategy used in the indirect methods one has to look for a fluorescent
species whose fluorescence is quenched by the analyte. In such a case the fluorescence
intensity of the target molecule is measured as a function of the concentration of the
analyte. For example, halide ions quench the fluorescence of quinine. Their
concentration can be measured by the quenching method. Another interesting example
is the common fluorescence quencher, O2. This paramagnetic species can also be
determined by the quenching method. The quenching method, however, has a
limitation. It will be useful only when the analyte is the only species that will cause
quenching of the fluorescent molecule, but it is little unlikely.

6.3

FLUORESCENCE SPECTROSCOPY IN
QUANTITATIVE ANALYSIS

You have learnt earlier that spectrophotometry is a good tool for the determination of
concentration of the analyte. However, at much lower concentration (ng level) of the
substances, the fluorescence spectrometry scores over spectrophotometry. It has come
to the fore front in utility because of higher sensitivity of the fluorimetric
determinations in comparison with usual UV-visible spectrophotometry. Further,
fluorimetry is more selective than UV-visible absorption spectrometry. It is more
selective on two counts. First, a number of molecules absorb strongly in the UV or
visible range but do not exhibit detectable fluorescence. Secondly, in case of
fluorescence two wavelengths (excitation and emission) are available whereas only a
single wavelength is available in spectrophotometry. Two samples having similar
absorption spectra may be distinguished if they fluoresce at different wavelengths.
Similarly, two analyte molecules having similar fluorescence spectra may be
differentiated by proper choice of excitation wavelength (selective excitation).
In addition, the life time measurements in fluorescence are very useful for the
determination of the analyte. The fluorimetric methods are preferred over the
spectrophotometric methods if the concentration of the material to be analysed is too
small; as small as 0.1 ng of analyte in 10 ml solution can be conveniently analysed by
fluorimetry. The selectivity of fluorimetry, however, is limited by the broad spectra
without much finer details. Further, the positions of bands are not sensitive to the
molecular structural details. Therefore, fluorimetry is not generally useful for
molecular identification.
Despite the fact that very few fluorescence species are in existence, the fluorimetric
determinations have their own place. The knowledge of excitation and emission
wavelength of the analyte facilitates the quantitative analysis. Most of the organic
compounds and metal chelates are usually analysed in the UV-Vis region of 200 800

30

nm with appropriate excitation wavelengths. Let us learn about the basic principle
behind the quantitative applications and also the factors that affect such
determinations.

6.3.1

Applications of
Fluorimetry and
Phosphorimetry

Concentration Dependence of Fluorescence

A spectrometer method can be put to quantitative analytical application only if the

spectral characteristics like position, intensity of the width of the spectral band can be
related to the concentration of the analyte. Let us establish a relationship between the
fluorescence intensity and the concentration of the analyte. You would recall from the
previous unit that, the fluorescence quantum efficiency, f , is defined by the
following expression.
f =

=>

No. of photons emitted

Intensity of fluorescen ce
=
No. of photons absorbed
Intensity of absorption
f =

Pf
Pa

(6.1)

On rearranging this equation we get, Pf = f Pa

As Pa = P0 Pt , we may write,
Pf = f ( P0 Pt ) = f P0 (1

Pt

P0

(6.2)

where,
Po = incident radiant power,
Pt = radiant power of emission spectra,
Pf = fluorescence power, and
f = quantum yield of fluorescence.
As Pt = P0 e bc
=> Pf = t P0 [1 e bc ]
You know that,

e x =1

x x 2 x3 x 4
+
+ ........
1! 2! 3! 4!

Substituting the expansion term in the bracket of the above equation and simplifying,
we get the following.
Pf = f P0 bc (1

bc
2!

(bc)2
)
3!

(6.3)

This, under the conditions of bc = 0.05, (i.e., by ignoring higher terms) gets
simplified to the following.
Pf = f P0 bc

(6.4)

According to Eq. 6.4, the measured fluorescence signal is 0 if the analyte

concentration is 0. The signal is a small number for low concentration. Therefore, for
low analyte concentration, the fluorescence measurement entails distinguishing a small
signal from no signal. Contrast it to the absorption spectroscopy where we need to
measure a small difference between two large numbers; I and I0. The detection limit in

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Molecular Spectroscopic
Methods-II

In actual situations, when

the analyte concentration
is zero, the observed
signal is not exactly zero.
This is due to the
background signals from
fluorescence of other
constituents of the
sample or contaminants
in the solvent or sample
cell. Therefore, we can
achieve low limits of
detection in fluorimetry
only if sufficient care is
taken to minimise the
background signals.

the most favorable cases rarely exceeds 10 8 moles. Whereas, in fluorescence

measurements under ideal conditions, the concentrations of the order of 1012 moles
can be measured.
Keeping all other factors except concentration constant in Eq. 6.4, the final equation
comes out to be:
Pf c

... (6.5)

Thus, the fluorescence intensity of a sample can be used for the concentration
determination of the analyte.

6.3.2 Factors Affecting Quantitative Applications of Fluorimetry

Eq. 6.5 may appear to be suggesting that the concentration determination using
fluorescence is quite straight forward. However, the following factors act as problem
areas in the course of such quantitative analysis.
Self quenching

You would recall from the previous unit that the fluorinating molecules may lose their
energy to other molecules through radiationless energy transfer during the collisions.
This leads to the quenching of fluorescence. You would also recall that the presence of
paramagnetic species like oxygen also cause quenching and thus proves to be of
hindrance in quantitative determination. Accordingly, the sample for fluorescence
determination needs to be flushed with nitrogen.
Radiant energy absorption

At higher concentrations of the analyte, as the radiation is absorbed by the analyte, the
successive layers of the sample get progressively reduced intensity of the incident
radiation. This reduces the signal of fluorescence. However, this problem can be
eliminated by dilution of solution or adopting standard addition method. You would
recall that we discussed about the standard addition method in Unit 2 (Sec. 2.6.3) on
UV visible spectrometry.
Self absorption

Another mechanism causing the deviation from the linear dependence on

concentration at high concentrations of the analyte is self absorption. Some analyte
molecules in the sample absorb the radiation emitted by other analyte molecules
leading to the decrease in the fluorescence intensity. In such situation also diluting the
sample proves useful.
Interfering species

In some fluorimetric determinations we may have some species, whose absorption

band overlaps with the absorption or emission bands of the analyte. In such cases,
these species affect the fluorescence emission intensity.
In recent years, there has been a growing need for developing highly sensitive and
selective probes for the detection of metal ions in environmental and biological
samples. A variety of divalent metal ions are known to be involved in the structural,
catalytic, and regulatory aspects of the biological system, and some such metal ions
serve as prognostics of certain human diseases. For example, Cu2+, Zn2+, and Fe2+ have
been found to be involved in aggregating -amyloid peptides during the onset of
Alzheimers disease. In addition, analysis of different biological samples including the
body fluids, etc. is crucial for diagnostic purposes and to plan a suitable treatment
strategy. It, therefore, becomes pertinent that we develop suitable analytical methods
for the analytical determination of the metal ions and other species in environmental

32

and biological samples. Let us take up the role of fluorimetry in environmental

monitoring.

Applications of
Fluorimetry and
Phosphorimetry

SAQ 1
List the factors adversely affecting the quantitative determinations of the analyte using
fluorescence spectrometry.
...
...
...
...
...
...

6.4

FLUORIMETRY AND ENVIRONMENTAL

MONITORING

In recent past the fluorescence measurements have found applications in diverse

fields; environment being one of the significant ones. In fact, in the air pollution
monitoring especially for analysis of NO NOx as well as SO2 gases no other method
is as good as fluorescence. Even in water pollution studies, the analysis of metal
pollutants like Be, Zn, V, S is best done by fluorescence methods. In order to
understand the implications of fluorescence measurements in air pollution monitoring
we would consider two types of important gaseous pollutants and their monitoring.

6.4.1

Analysis of Gaseous Pollutants

The analysis of gaseous pollutants is based on the phenomenon of chemiluminescence.

We begin with the determination of NO NO2 as atmospheric pollutants. The
principle of such a determination is discussed here. The procedural details can be
found in the laboratory manuals or from the manuals of pollution control agencies.
Determination of NO NO2

In order to determine the amount of NO, the gas is passed through the reactor in which
it reacts with ozone. Initially the excited NO* species is produced as per the reaction
2

given below.
NO + O3
NO*2 + O2
The activated NO*2 then gives chemiluminescence broadband in the visible to infrared
range (600 2800 nm) and reverts back to a lower energy state. The emitted photons
are proportional to the amount of NO present and are measured with the help of a
photomultiplier tube (PMT).
NO2 + h (chemiluminescence)
NO*2
M

NO2 (M = N2, O2, H2O, etc.)

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The presence of other species, M contributes towards nonradiative deactivation. The

intensity of the emitted radiation (I) is given by the following equation:

I = Constant [NO] [O3] [M]1

The luminescence intensity is found to be directly proportioned to the concentration of
MO, O3 and is inversely related to the other species, M.
As [M] pressure employed, at low pressures,

I=

d ( photons )
[O 3 ][ NO]
dt

At the operating pressure of 0.01 0.05 atmosphere the intensity of the signal is quite
good and the extent of signal from PMT is proportional to concentration of NO and
the concentrations as small as 1 ppb of the gas can be measured.

Fig. 6.1: Schematic layout for the determination of NO-NO2 by chemiluminescence

method

In order to determine the amount of nitrogen dioxide, NO2, in a sample having no

nitric oxide, NO, it is first converted to nitric oxide, NO, by passing through a catalytic
converter. Thereafter, it is passed through the reactor for activation by ozone as
discussed. The photon count from the PMT is proportional to the concentration of NO
which is a measure of NO2 before it was converted to NO.
In the sample containing a mixture of NO and NO2, the sample is passed through the
reactor and the amount of NO is determined. Thereafter, in a separate experiment the
sample is passed through the converter (so as to convert the NO2 to NO) before
sending to the reactor. The photon count in such a case gives the total amount of NO
and NO2 in the air sample. The amount of NO2 is obtained by subtracting the value for
NO from that of the mixture.
Determination of SO2

As you know, sulphur dioxide is one of the main components of air pollution in many
parts of the world. The combustion of sulphur containing fossil fuel for domestic use
and power generation along with not so well controlled combustion of the fossil fuels
in industrial installations are globally the chief contributors to the increased
environmental levels of SO2. This increased concentration has serious effect on the
human health as well as the ecological system. Therefore, monitoring and control of
the SO2 concentration in the environment is of paramount importance. It is pertinent,
therefore, to have reliable methods for the determination of SO2 in the environmental
samples.
There are several good methods for monitoring of SO2 in the air samples. In the
commonly employed West Geake method, the air is bubbled through 0.1 M sodium
tetrachloromercurate solution to obtain stable, non-volatile dichlorosulphitomercurate.

34

[HgCl4]2 + 2SO2

[Hg(SO3)2]2 + 4Cl + 4H+

The complex is made to react with pararosaniline and formaldehyde to form the
intensely coloured para-rosanilinemethylsulphonic acid. The absorbance of the
solution is measured by means of a suitable spectrophotometer. Concentration of
sulphur dioxide in the range of 25 1050 g /m3 can be measured under these
conditions.

Applications of
Fluorimetry and
Phosphorimetry

In the fluorimetric method, a pulsed UV source is employed to excite the SO2

molecules. These excited molecules emit a characteristic fluorescence with intensity
proportional to the concentration of SO2 .
SO 2 + h SO*2
SO*2 SO 2 + h (fluorescen ce)

The fluorescence occurs at low pressure (0.5 atm). The fluorescence signal is detected
by a photomultiplier tube and is suitably recorded. One can measure 0.5 1000 ppm
of SO2 by fluorimetry.
The reaction with para-rosaniline and formaldehyde can be shown as below.
NH 2

NH2

+
NH2

SO 2 +

HCHO

NH 2

+
C

NH

C SO 3
H2

NH 2

p - Rosaniline

p - Rosanilinemethylsulphonic acid

Another method, based on chemiluminescence is even more reliable, accurate and less
time consuming. In this method, the sample containing atmospheric sulphur
compounds like, sulphur dioxide, hydrogen sulphide and merceptans, etc. is
combusted in the hydrogen flame. This generates a sulphur dimer that decomposes
with the emission of characteristic blue light with max at 384 nm and 394 nm. One can
detect as small as 5 ppb of SO2 gas by this method; the reactions for SO2 gas are as
follows:
4 H 2 + 2SO 2
S*2

S*2 + 4H 2

S2 + h

The chemiluminescence method is rapid and permits analysis of microgram

concentration of pollutants. The methods have been commercialised and one can buy
automated analysers from the market. The method is free from interferences by CO2,
CO, SO2, O3, hydrocarbons and water vapour. It provides continuous measuring
devise. As regards the limitations the problem area is that all nitrogen containing
compounds produce NO2 on combustion. It is important to know concentration ranges
of SO2 as well as NO2 in the atmosphere (Table 6.1).

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Table 6.1: Atmospheric level of SO2 and NO2

Conc.

SO2
NO2

Concentration (ppb)
Source vicinity
Urban

Source
2 106

10

106

103

Rural

Remote

50- 100

5-50

20-500

5-50

6.4.2 Analysis of Water Pollutants

Let us now consider the analysis of metal pollutants like Al, Zn, and the anion, F in
the aquatic environment by fluorescence methods.
Analysis of Aluminium

Aluminium is used as coagulating agent in the process of purification of polluted

water. Due to its toxic effects on living beings, Al represents an environmental hazard,
particularly under increased acidic conditions. The growing concern over the presence
of increased Al concentrations in soil solutions and fresh waters has resulted in the
development of numerous analytical techniques for the determination of Al species in
water. As the concentration of aluminium in the water sample may be very small we,
therefore, need a sensitive method for its analysis. Fluorescence measurements provide
for such a method.
Aluminium forms chelates with a wide variety of chelating agents, like, acid alizarin
red, solochrome dark blue, morin (pentahydroxyflavanol), and 8- hydroxyquinoline
etc. However, the most commonly used fluorinating agents for aluminium are morin
and oxine. At a pH of 3.3, aluminium forms complex with morin which shows
maxima at 430 nm (for excitation) and at 500 nm (for emission wavelength). As small
as 0.001 g of Al can be analysed by this method. However, F and Th4+ ions etc.
show interference.
In the second method involving use of 8-hydroxyquinoline complex, aluminium tris
(8-hydroxyquinoline) is obtained at pH 6.7. The complex is extracted with chloroform
and has excitation maxima at 350 nm while emission wavelength is 520 nm. Only
gallium shows interference in this method and the sensitivity is of the order of
0.1g / ml.
The structures of morin and 8-hydroxyquinoline as the common fluorinating agents
are given below.
2'

HO

HO

4' OH

O
2
5
OH

3 OH
O

Morin

OH
8-Hydroxyquinoline

Analysis of Zinc
The extensive utilisation of Zn in the process of galvanisation leads to considerable
amount of Zn in the drinking water and warrants a suitable method for its analysis.
The most favored fluorimetric method for the analysis of zinc also involves the use of

36

the oxine i.e. 8-hydroxyquinoline with acetate buffer at pH 6.0, the complex is
obtained. In acetate buffer having a pH of 6 the complex is extracted in chloroform
and has excitation wavelength of 420 nm. However, Al, Mg and Fe interfere in this
method.

Applications of
Fluorimetry and
Phosphorimetry

Analysis of Fluoride
Fluoride is important component of potable water. The growth of teeth especially in
children is impaired in the absence of fluoride however, an excess of fluoride in water
causes fluorosis. It, therefore, becomes pertinent to ascertain and control the amount of
fluoride in potable water.
Many methods based on substitution reaction are known for the determination of
fluoride ions. The most significant one is the one that involves the formation of a
ternary complex with zirconium and calcium blue. This method is relatively free from
interferences. The reaction occurs at a pH of 2.5 and the excitation is at 350 nm with
an emission of 410 nm. The method is quite rapid and has sensitivity in the ppb range.
Another method of analysis involves quenching fluorescence intensity of Zr-alizarin
complex. The quenching of fluorescence is directly proportional to the concentration
of fluoride in water. Zr-alizarin complex is obtained at a pH 4.6 and has the excitation
and emission wavelengths as 470 and 520 nm, respectively. As small as 0.001 g/ml
of fluoride can be analysed by this method, however, several metals like Be, CO, Cr,
Cu, Fe, Ni, Th, and PO 34 ions show very strong interference and they must be
removed before fluorescence measurement.

SAQ 2
What is the role of ozone in the analysis of NO-NO2 by chemiluminescence method?
...
...
...
...
...
...

6.5

FLUORESCENCE SPECTROSCOPY IN MEDICINE

AND BIOLOGY

The use of fluorimetry in the fields of medicine and biological samples is well
established. In fact, fluorescence technique has got a big boost with its applications in
Biological sciences. Today, highly selective and sensitive biochemical determinations
can be accomplished by fluorimetry. Spectrofluorimeters tailored to suit the need of
specific sample to be analysed are available in the market. Interphasing fluorimeter
with chromatograph has also yielded good results, as it facilitates analysis of mixtures
without resorting to tedious separations.

Fluorimetry is more
sensitive and selective
than usual
spectrophotometry.

In clinical situations many a times we need quick analysis of the clinical samples as
the diagnosis and treatment options depend on the outcome of such determinations.
For example, the enzyme released after heart attack must be analysed within 15
minutes, so as to ascertain the future course of action. In these life saving situations
the fluorescence methods find favour as these are simple, rapid, selective and
sensitive.

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The cleanup, separation and determination of the analyte are the important steps
involved in the analysis of clinical samples. For example, the removal of red blood
cells from blood is necessary before one undertakes fluorescence analysis. In some of
the biological samples, we need to undertake deproteination as the latter quenches
fluorescence. In case a direct determination of the analyte faces too many interference
it is advisable to use a suitable agent and convert the analyte into a fluorinating
derivative. Therefore, many nonfluorescent compounds are converted into fluorinating
derivatives prior to their analytical determination.
Fluorescence and phosphorescence are particularly useful in physical Biochemistry
since they provide much information with regard to the interaction of molecular
complexes with their environment. Molecular rotation and reorientation in
biochemical systems can be conveniently studied as they have similar lifetimes to the
excited singlet and triplet states.
Let us take up some common applications of fluorimetric analysis of biological
samples.

6.5.1 Analysis of Amino Acids and Proteins

Quantification of total protein content in a sample is common to many applications in
basic science and clinical research. Over the years, many different absorbance based
colorimetric methods to quantify protein have been developed. These methods work
well however, these are subject to interference by many compounds. Several methods
based on fluorescence measurements have been developed for protein estimation.
Fluorescamine and o-phthalaldehyde have been used with success to quantify protein
content of samples.
As regards the protein samples, three amino acids constituting them exhibit natural
fluorescence. These are tyrosine, tryptophan and phenylalanine; other amino acids
however, need a suitable agent to convert them to fluorescent derivatives.
Fluorescamine, a heterocyclic dione, is very useful agent for the analysis of the non
fluorescent amino acids. It reacts with the primary amino group of the amino acid to
form a fluorescent product. The fluorescence of a solution containing protein and
fluorescamine is found to be proportional to the quantity of free amino groups present.
Therefore this reaction forms the basis of fluorimetric assay of the proteins and amino
acids. Fluorescamine has also been used in labelling casein-the milk protein, so that it
can be used as a substrate for measuring protease activity.

O
O

R-NH2

O
OH

O
Fluorescamine

Femto : 1015

38

Another very important reagent is o-phthalaldehyde (OPA). It is probably the most

widely used reagents and has sensitivities in the femtomole range. The reagent reacts
with the primary amino group at alkaline pH ranges in the presence of certain thiols
such as mercaptoethanol or ethanethiol. The reaction is quite quick and is essentially
complete within a minute.

Applications of
Fluorimetry and
Phosphorimetry

O
SC2H5
CH
+

C2H5SH

R-NH2

CH
O
o-Phthalaldehyde

6.5.2

Fluorimetric Determination of Blood Glucose

As you are aware, the analysis of blood glucose is most critical for diagnosis of
diabetic patients. The people with diabetes mellitus need to constantly monitor their
blood glucose levels in order to detect and control fluctuations in glucose level that
could lead to hyperglycemia (high blood glucose levels) or hypoglycemia (low blood
glucose levels). It is, therefore, necessary to have a simple, specific and rapid test for
the concentration of glucose in the blood. Here again fluorescence method can be
exploited.
Nowadays, blood glucose levels are measured by a procedure based upon the enzyme
named glucose oxidase. Since an enzyme is used, it is specific for only D- glucose,
and will not be subject to interferences from other molecules in the blood. The
determination of blood glucose is based on the following reactions.

The concentration of the

glucose is related to the
intensity of colour
produced.

Step 1: The enzyme glucose oxidase catalyses the oxidation of -D-glucose to form
D-glucono-1,5-lactone and hydrogen peroxide.

- D - Glucose + O2

glucose oxidase

D - Glucono - 1,5 - lactone + H2O2

Step 2: D-glucono-1, 5-lactone then spontaneously gets hydrolysed to produce

gluconic acid.
D-Glucono-1,5 -lactone + H2O

D-Gluconic acid

As the -D-glucose is rapidly converted to the beta form, therefore, all of the glucose
is measured at one time. This glucose oxidase catalysed reaction can be monitored by
fluorescent detection of the consumption of oxygen or by monitoring the production of
hydrogen peroxide. The amount of hydrogen peroxide produced is determined by
using a fluorophore named luminol.

H2O2

+
C
NH2

Luminol

NH
NH

base
catalyst

C
NH2

O
O

3-Aminophthalate*

C
NH2

O
O

In spectrophotometric
determination hydrogen
peroxide is made to react
with o-toluidine or
2-methylaniline in presence
of the enzyme peroxidase
to produce a coloured
species.

3-Aminophthalate

The mechanism of fluorescence emission by the dianion formed above is as follows:

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NH2

NH2

O
*
O
O

Intersystem
crossing

+ N2

During clinical analysis

the stabilisation of
reagents and time factor
is very valuable. For
rapid analysis in clinical
chemistry reagents must
be kept ready to use.

Triplet dianion (T1)

Excited State

NH2

*
O

O
Singlet dianion (S1)
Excited State

hv

O
Ground State dianion (S0)

The detection limit of glucose by this method is 50 g /ml. This reaction can be used
for analysis of fructose or sucrose also. Nowadays some biosensors are also being
developed to measure blood glucose levels. These biosensors are based on sensitive
fluorescence measurements which work by monitoring changes in the intrinsic FAD
(flavin adenine dinucleotide) fluorescence of glucose oxidase. FAD is the cofactor of
the enzyme.

6.5.3 Analysis of Blood Serum

In order to facilitate diagnosis, the serum from human blood is analysed for the
presence of different ions or metabolites. Here too fluorescence measurement plays an
important role and the leukocyte in blood are analysed. For this excitation and
emission wavelength are 465 nm and 475 nm, respectively.

6.5.4 Analysis of Creatinine Phosphokinase

Creatinine phosphokinase (CPK) is an important enzyme whose level in the body
increases after heart attack. The fluorimetric method of determination of CPK is based
on the following reactions.
Creatinephosphate + ADP Creatine + ATP
ATP + glucose ADP + Glucose 6-phosphate
Glucose 6-phosphate + NAD 6-phosphogluconate + NADH + H+
wherein, NAD is nicotinamide adenine dinucleotide. The fluorescent intensity is
measured at 450 nm. Similarly, Galactose has been determined in blood and plasma
using a continuous flow system and two coupled enzyme systems, namely, galactose
oxidase and peroxidase.

6.5.5 Analysis of Calcium Ion

Calcium ion in biological systems is determined fluorimetrically either by titration
with EDTA using calcein as indicator or by direct estimation using calcein as the
reagent. The second method is based on complex formation between calcein and
calcium ions in alkaline medium. The fluorophore so formed has an excitation
wavelength of 450 nm and emission is at 520 nm. Other cations are found to be
noninterfering.
Several interesting biochemical analysis are done by combining the chromatographic
separation and fluorimetry. The chromatographic methods coupled with fluorimetry
use a suitably developing fluorophore. The HPLC separations at room temperature
are preferred. For example, a variety of indols in biological fluids are analysed by
fluorimetry. The excitation is at 292 nm and emission occurs at 330 nm. Table 6.2
gives a list of important organic molecules present in biological samples and their
fluorescence characteristics.

40

Table 6.2: The fluorescence characteristics of organic molecules of biochemical

origin
S.

Compound

Solvent

pH

No.

excitation
(nm)

emission
(nm)

Sensitivity

1.

Brucine

H2O

305

500

Good

2.

Codeine

H2O

285

350

Poor

3.

Fluorene

Pentane

300

321

Good

4.

Hippuric acid

H2SO4

270

370

Good

5.

Indoles

H2O

315

350

Fair

6.

LSD

Acid

325

445

Good

7.

Morphine

H2O

285

350

Poor

8.

Procaine

H2O

11

275

345

Fair(0.01)

9.

Penicillin

Derivative 1M HCl

365

540

Good

10.

Nicotinamide

CNBr

7.0

365

470

Reasonable

11.

Folic acid

H2O

7-11

490

515

Fair

12.

Ascorbic acid

Derivative

7.5

365

Blue
white

6.5.6

Applications of
Fluorimetry and
Phosphorimetry

Good

Bioluminescence

In the previous unit, you read about the origin of chemiluminescence as a consequence
of a chemical reaction. You have learnt about some environmental applications of
chemiluminescence in section 6.3. Measurement of light from a chemical reaction is
highly useful because the concentration of an unknown can be inferred from the rate at
which light is emitted. The rate of light output is directly related to the amount of light
emitted and accordingly, proportional to the concentration of the luminescent material
present. Therefore, light measurement is a relative indicator of the amount of
luminescent material present in the sample of interest. The oxidation of luminol in
alkaline solution is a well known example of chemiluminescence.
The phenomenon of luminescence occurring in living systems or the compounds
extracted from the living systems is called the bioluminescence. Let us try to
understand this phenomenon and its significance in analysis.
You might have seen a firefly and may also would have come across flashing fish,
glinting glow worms, etc. and would have wondered about the why and how of the
glow. Some natural purposes of such a glow include, attracting a mate, attracting prey,
camouflage, deterring predators, and aiding in hunting, etc. As regards the how or
origin of such a glow, it is due to the result of biological processes that lead to the
emission of light leading to the glow. These processes involve the use of a chemical

41

Molecular Spectroscopic
Methods-II

called luciferin and an enzyme called luciferase. As mentioned above, this

phenomenon of emission of light by a living creature or compound extracted from
living systems is called bioluminescence.
The reactions involved in the process of bioluminescence are given below.
LH2 + E + ATP + Mg2+ E-LH2 AMP + Mg Ppi
E-LH2 AMP + O2 AMP + CO2 + H2O + Oxyluciferrin*
Oxyleuciferrin* Oxyluciferrin + h
In above example E = enzyme luciferase, LH2 = substrate luciferin, AMP = adenosine
monophophate and PPi = pyrophosphate. The oxyleuciferrin -a fluorencent radiating
compound is generated due to enzyme reaction. ATP can be indirectly analysed.
In the first step the firefly enzyme luciferase catalyses the formation of a complex
between luciferin and ATP. The complex is called luciferyl adenylate is oxidised by
oxygen, leading to the production of a cyclic peroxide that eventually becomes highenergy oxyluciferin. The oxyluciferin in an excited state relaxes back to the ground
state, accompanied by the emission of light.
S

OH
O

Luciferin

The light output is directly proportional to the concentration of the limiting reactant in
the system. In the ATP/ luciferin-luciferase system, when the total sample volume is
held constant and ATP is the limiting reactant, the light output is proportional to the
ATP concentration. When luciferase is the limiting reactant, the light output is found
to be proportional to the luciferase concentration.
The Bioluminescence phenomenon is being exploited for research in the medical field.
Certain bioluminescent bacteria are being used to follow the progression of infection
in mice. The spread of infection can be gauged and the effect of different antibiotics
can be followed visually. In addition, the bioluminescent organisms are being used to
trace the ATP and calcium in the cell, and to assist in AIDS research besides other
applications.

SAQ 3
What is the role of H2O2 in analysis of glucose by fluorimetry?
...
...
...
...

6.6

FLUORIMETRIC ANALYSIS OF INORGANIC

SUBSTANCES

Several minerals and alloys contain metals and many of such metals are analysed by
fluorescence spectroscopy. In view of emphasis on the fluorimetric analysis of

42

elements, in this section we will take an overview of analysis of inorganic compounds

including metals, non-metals, minerals and alloys by fluorescence spectroscopy.
While fluorescence spectroscopy is widely used in the qualitative and quantitative
analysis of inorganic compounds; phosphorescence spectroscopy does not find many
applications. Phosphorescence measurement is used mainly in the area of inorganic
phosphors. Further, though several inorganic substances are fluorescent or
phosphorescent in the solid state, the majority of analyses are performed in solution
phase.

Applications of
Fluorimetry and
Phosphorimetry

Aluminium was the first element to be analysed by fluorimetry using morin reagent.
Today, a large number of ions are being analysed using fluorescence; the number of
cations analysed being much larger as compared to anions.
The analytical determinations of inorganic species by fluorimetric methods involve
different types of chemical reactions or methodologies. Before taking up the
applications of fluorimetry to inorganic substances let us learn about these reactions.

6.6.1

Chemical Reactions Producing Fluorescence

There are several kinds of reactions which lead to generation of fluorescence. These
are binary or ternary complexation (ion association), substitution reactions, redox
reactions, enzymatic reactions, kinetic methods, extractions, etc. We would consider
briefly each of these methods with examples to illustrate the principle.
Binary Complexation

A binary complex contains one central ion and one ligand only e.g., Al combines with
alizarin to produce fluorescent binary complex. Similarly, several derivatives of
8-hydroxyquinoline, azo dyes, Schiffs bases, etc., show fluorescence by the formation
of binary complexes with metal ions.
Ternary Complexation

In a ternary complex formation, a central ion is coupled with two ligands. These
complexes are called as ion association complex e.g., interaction of Hg or Sn with
Rhodamine in the presence of chloride or bromide ions.
Substitution Reactions

In some cases an anion is made to react with cation (central atom) of a metal-organic
compound complex. This undergoes a substitution reaction and as a result, the organic
ligand is set free. The analysis of F , CN , or sulphide ions is based on such
substitution reactions. Some ions are determined by quenching or generation of non
fluorescence organic cation complex.
Redox Reactions

In this case the reaction is between an organic reagent and the inorganic species. The
anions like Br , PO43 or cations like Ce (IV), Fe (III), Hg (II), and V (V) are analysed
by these kinds of reactions (Table 6.3). For example, chloramine T and nicotinamide
react with cyanide to give fluorinating cyanogen chloride.
Enzymatic Reactions

Some fluorimetric determinations involve the use of enzymes. The inorganic species
reacts with enzymes but the methods are not selective. For example, in the analysis of
arsenic, the enzyme glyceraldehyde-3-phosphate dehydrogenase is used.

43

Molecular Spectroscopic
Methods-II

Kinetic Reactions

Some reactions need heating, therefore, are catagorised under the kinetic methods.
Fort example, aluminium reacts with oxine or its sulpho derivative to form fluorescent
complex, the initial rate being directly proportional to concentration of the aluminium
species.
Extractive Fluorimetry

In many fluorimetric determinations the interferences are too serious to be ignored.

These interfering species need to be removed before taking up the determination. The
ion to be determined is extracted out of the sample, leaving the interferences behind.
Solvent extraction is one such method that facilitates the removal of interferences e.g.
Tl (III) is first extracted in benzene with crystal violet which in turn is substituted by
butyl Rhodamine B. Like extractive photometry which is so extensively used in
analytical chemistry, extractive fluorimetry is getting very popular e.g. Bi(III) is
analysed with dibenzoylmethane at 1g level or niobium (Nb) with oxine can be
determined at 0.18 ppb level. Beryllium can be analysed by dibenzoylmethane with
pyridine and as small as 0.0004 g/ml of Be can be conveniently analysed by
extractive fluorimetry.
Having learnt about different types of reactions that aid in the fluorimetric
determination we can now take up the applications of fluorimetric determinations of
inorganic substances. In a broad way, the applications of fluorescence in the analysis
of inorganic species have been grouped under three heads as given below.

Inorganic substances showing luminescence

Fluorescence with inorganic reagents

Fluorescence with organic reagents

The first group pertains to direct analysis whereas the other two exploit indirect
methods. Let us begin with inorganic substances having intrinsic fluorescence i.e. they
are fluorescent in nature.

6.6.2 Inorganic Substances Showing Luminescence

As mentioned earlier, only lanthanides and uranium compounds show fluorescence in
solution hence these ions are analysed directly without use of any of the organic or
inorganic reagents. Within lanthanides Ce, Pr, Nd show fluorescence with electronic
transitions from 5d to 4f shells e.g. for Ce (III) ex. = 260nm, em. = 350 nm. In
dilute solutions, the spectrum is very sharp due to transitions of f-electrons. The
uranium compounds show natural fluorescence at 520-620nm. U (VI) is extracted with
tributylphosphate and is back washed with Na3PO4 and the resulting fluorescence is
measured. In presence of H3PO4, the intensity of fluorescence is enhanced.

6.6.3 Fluorescence with Inorganic Reagents

Most of the metals show fluorescence in the presence of suitable reagents. In other
words, these can be analysed by the indirect method. For example, certain metals like,
Tl, Sn, Pb, As, Sb, Bi, can show fluorescence at low temperature on reaction with
acids like, HCl, HBr. Most of the p- Block elements in presence of HCl/HBr show
fluorescence with excitation wavelength in ultraviolet and emission wavelength in the
visible region. For example, arsenic has ex. = 350 nm and em. = 690 nm. The
detection limit varies from 0.002-1.0 ppm. Apart from acids, Na2WO4 promotes
fluorescence for lanthanides at pH = 4.5. The analysis of UO2 (SO4) in H3PO4 by Tl (I)
ions in the range of 0.1-80 ppm is done with fluorescence quenching. Unfortunately
several of the ions show strong interference and reduce fluorescence intensity. Some

44

Applications of
Fluorimetry and
Phosphorimetry

metals and the reagents used for the determination along with the spectral
characteristics, sensitivity and the interferences are given in Table 6.3.
Table 6.3: Fluorimetric determination of inorganic materials
S.No Metal

Reagent

Condition

ex

em

Sensitivity

Interferences/
Remarks

1.

Ca(II)

Calcein

0.4 M KOH

360

485

0.02

Ba, Sr

2.

Ga(III)

Oxine (pH 2.6)

CHCl3 Extract. 436

470

0.005

Cu, Fe, Mo, V

3.

In(III)

Oxine (pH 5.1)

CHCl3 Extract. 365

535

0.04

Al, Be, Cu, Fe

4.

Mg(II)

Oxine

H2O

365

440

0.1

Ca ( to be masked)

5.

Sn (IV)

Morin

Hexane

415

420

0.001

Extraction, no
interference.

6.

Th (IV)

Morin

0.01M HCl

420

520

0.02

Al, Ca, Fe, Zr

7.

Tl (III)

Rhodamine

2 M HCl

360

0.01

Extraction in benzene

8.

U(VI)

NaF

Conc. H2SO4

254

Yellow
green

0.1

Many interfere

9.

Zn (II)

Oxine

pH 9.5

420

Green
Yellow

1.0

Al, Fe, Mg

10.

Zr (IV)

Morin

2 M HCl

425

515

0.02

Al, Sb, Sn, Th, U

6.6.4

Fluorescence with Organic Reagents

The inorganic substances may be analysed using organic reagents for the complex
formation. The formation of fluorescent complex between the organic compound and
the metal ion can be exploited in two ways. These may readily be used for the
determination of metal species using organic reagent. Alternatively, the organic
compound may be determined using metal ion as the reagent. These are called
fluorescent probes. However, the former has far more applications than the later. In
fact the later is currently an important research area.

Many nonfluorescent
organic compounds show
fluorescence on forming
a complex with a metal
ion.

The most effective organic reagents are the ones that can form a chelate or a ring with
the metal ion by binding with it at more than one place. This in turn requires that the
organic compound has two or more functional groups. Further, these functional groups
should be so placed in the molecule that on chelation they form 5- or 6-membered
ring. Table 6.4 gives a list of organic reagents that form fluorescent complexes with
metal ions, the types of the complexes formed and the detection limits.

45

Molecular Spectroscopic
Methods-II

Table 6.4: Organic reagents producing fluorescence

Common
Metals

Reagents

Lower limit of
Type of reactions
producing fluorescence level (ppm)

Ag 2+

Eosin

Ternary complex

8 10 2

Al3+

Morin

Binary complex

2.5 10 4

Be2+

Morin

Binary complex

1.6 10

Cd2+

Calcein

Binary complex

5 10 2

Co3+

1-(2-pyridylazo)-2naphthol (PAN)

Binary complex

5.9 10 2

Cu2+

Bathocuporine

Quenching

6 10 3

Fe3+

Rhodamine B

Quenching

2 10 3

Hg2+

Thiamine

Redox reaction

5 10 1

K+

Eosin + 18 Crown 6

Ternary complex

1 10 2

Mg2+

Oxine

Binary complex

1 10 2

Mn2+

1-10 phenanthrene +
Eosin

Ternary complex

1 10 1

Mo6+

Morin

Binary complex

9 10 1

Ni2+

Na(PAN)

Quenching

6 10 5

Pb2+

Eosin

Quenching

1.5 10 1

Sb3+

Rhodamine 6G+Cl-

Ternary complex

2 10 1

Sn4+

Marine

Binary complex

1 10 1

Zn2+

8-Hydroxyquinoline

Binary complex

4 10 1

Alizarin

Quenching

While Mg, Cd, Zr, Zn, are analysed by complex formation with organic reagent, few
of the metals can be analysed by methods based on phenomena of quenching; the
most favoured metals being Ga, Al and Be (Table 6.4).
The transition elements rarely form fluorescent chelates however, copper is one
exception to this rule. Amongst anions borate has maximum number of methods
available for analysis. Then we have fluoride and to some extent sulphide anion which
is generally analysed by substitution reaction.

46

6.7

FLUORESCENCE AND MINERAL ANALYSIS

Several minerals like calcite, fluorite, rubies and zircon on exposure to UV radiation
start emitting fluorescence. The only requirement being that the substances inhibiting
and quenching fluorescence must be absent. e.g., gypsum (CaSO4 2H2O), one of the
common minerals in sediment environments exhibit red fluorescence on exposure to
UV radiation. Similarly, the phosphate mineral e.g. Zircon shows fluorescence on
exposure to UV radiation. The feldspar, autunite and scheelite the minerals
containing silicon, uranium and molybdenum respectively, show bright fluorescence.

Applications of
Fluorimetry and
Phosphorimetry

You would recall that the

term fluorescence was
coined on the basis of
fluorspar-the mineral
form of calcium fluoride
that is fluorescent

Fig. 6.2: Fluorescent mineral; a) Gypsum, and b) Adamite. Gypsum fluoresces under long
wavelength UV radiation whereas the columnar crystals of admit glow under
short wavelength UV radiation

Amongst fluorites the mineral willemite shows fluorescence in the present of S block
metals. Some crystalline materials absorb light in the UV region and emit in the
visible region. In fluorescent lamps, the ultra-violet radiation from low-pressure
mercury arc (Hg vapor emit light at 253 and 184 nm) is converted to visible light by
calcium halo-phosphate phosphor (Ca10F2P6O24). The crystalline materials emitting
fluorescence are refered to as crystallophosphors. Many vanadates oxyhalides, oxides,
etc. show fluorescence as matrices. Table 6.5 lists application of crystallophosphor for
analysis of metal ions.
Table 6.5: Applications of crystallophosphor for analysis of metal ions
Elements

Matrix

Determination in

Detection limit

Sm(III)

TbPO4

Tb4O7

10 4

Sm(III)

CaWO4 +Gd

Gd, N oxides

25-50 g

Eu (III)

CS2NaTb Cl6

Tb2O7

5 10 5

Mn (II)

Li Mn
tungsten ate

Water; HCl

0.01 mg

Cu(II)

Ag+ ZnS

Effluents

0.01-500ppm

Sb (V)

CaO

H2SO4+H+

1 10 4

Sn (IV)

KI

HCl

0.01mg

Pb (III)

NaCl + CaO

NaCl pellet

5-200ppb

Bi(III)

CaO

NaPO2 ,granite

0.1-30 g

47

Molecular Spectroscopic
Methods-II

Polycrystalline substances containing traces of ionic activators of luminescence are

also known. They contain crystalline imperfections; a large sized oxide ion being
predominant in them. Xenon lamp, lasers, cathode rays, x-rays are used for exposure.
The technique is mainly used for the analysis of lanthanide elements.

SAQ 4
Name two anions which are extensively studied by fluorimetry methods?
...
...
...
...

6.8

You would recall from the

previous unit that because
of long life-times, the
molecule has a very high
probability of losing its
excess energy by
radiationless relaxation and
as a result, phosphorescence
is not routinely observed in
solutions at room
temperature.

PHOSPHORIMETRIC METHODS IN CHEMICAL

ANALYSIS

You have learnt in the previous Unit that Phosphorescence has been observed from a
wide variety of compounds and is differentiated from fluorescence by the long-lived
emission of light after extinction of the excitation source. In comparison to the
fluorescence methods, the phosphorimetric technique is not much used for analytical
purpose. The applications of phosphorescence have been somewhat limited in the past
due to the lack of suitable instrumentation. Another impediment in the extensive usage
of phosphorescence is the practical difficulty in measuring the signal, because the
measurements are to be made at cryogenic temperatures. It is generally necessary to
freeze the sample, taken in special solvents, using liquid nitrogen.
The problem is further augmented by the fact that in comparison to fluorescence, the
phosphorescence life time is much larger whereby the molecule has a very high
probability of losing its excess energy by radiationless processes like, internal
conversion, bimolecular collision, and photodecomposition, etc. As a result,
phosphorescence is not routinely observed in solutions at room temperature. This is
measured in viscous media or from molecules adsorbed on solid surfaces where these
nonradiative processes are minimized or deactivated. Oxygen also promotes
radiationless deactivation of the triplet state and is effective in preventing
phosphorescence. Therefore, a thorough degassing of the solution is required before
measurement.
However with the introduction of new instrumental methods and the advances made in
room temperature phosphorescence, phosphorimetry is poised to make big leaps in the
domain of clinical chemistry and in the areas of forensic, environmental and
pharmaceutical sciences. Let us learn about room temperature phosphorescence.

6.8.1 Room Temperature Phosphorescence

New advances in analytical phosphorimetry have shown that phosphorescence
emissions can be obtained at room temperature in solution. The phosphorescence of
polyatomic aromatic compounds adsorbed on a variety of solid supports has been
observed at room temperature. The phosphorescence spectra obtained from polar
organic molecules adsorbed onto filter paper, silica and other chromatographic
supports have been found to be reasonable for analytical purposes. This phenomenon
of measuring phosphorescence at room temperature is refered to as room temperature
phosphorescence (RTP) and has opened newer areas to analytical application of
phosphorescence. It has been demonstrated that several analytes are able to give
room-temperature phosphorescence (RTP) in organized media such as micelles and
cyclodextrin solutions. Cyclodextrins (CDs) are cyclic oligosaccharides composed of

48

D-glucose residues obtained by (14) linkages. The three major cyclodextrins,

, , and -CDs, are formed by six, seven, and eight glucopyranose units, respectively.
The CDs provide a shielding environment to the excited species from quenchers and
nonradiative pathways.

Applications of
Fluorimetry and
Phosphorimetry

In cyclodextrins the monomers are coupled to form a rigid, conical structure with an
interior hydrophobic cavity as shown in Fig. 6.3 (a), and have a unique ability to form
stable inclusion complexes with a variety of molecules. Fig.6.3 (b) gives a schematic
representation of inclusion complex formed by phenanthrene in a cyclodextrin cavity.

(a)

(b)

Fig. 6.3: a) Formation of a cavity by cyclodextrin; and

b) Schematic representation of inclusion complex in a cyclodextrin cavity

The analytes in cyclodextrin cavities produce intense and well-structured

phosphorescence signals at nanomolar concentrations. The detection limits of
phenanthrene and acenaphthene - two typical phosphors in extremely sensitive RTP
measurement in cyclodextrin cavities are estimated to be of the order of 5 10 13 M
and 1 10 11 M, respectively.
Further, the presence of heavy atoms like, iodine, silver, lead, etc. in the sample or in
the solid matrix is found to improve the sensitivity of the technique and helps in the
analysis of complex mixtures by RTP. The presence of a heavy atom is an important
factor for RTP detection because this type of atom favors the intersystem crossing
from the singlet state to the triplet state of the guest. The analyte, the cyclodextrin or
other matrix and the component containing the heavy atom form a ternary complex
which provides adequate protection from quenching and constitutes a feasible
approach for enhancing the phosphorescence emission in solution. The inclusion of
the heavy atom in a separate molecule makes it possible to excite phosphorescence
from molecules containing only carbon, hydrogen, oxygen and nitrogen.
Recently, it has been reported that RTP can be observed in solutions without the use of
an organized medium. In this method, a significant amount of a heavy-atom salt like
potassium iodide and thallium (I) nitrate along with sodium sulfite is added to the
analyte. The sodium sulfite acts as an oxygen scavenger. The RTP emission is a
consequence of intermolecular protection and is refered to heavy-atom-induced RTP
(HAI-RTP).
The compounds showing RTP can be divided into two types. The first group includes
inorganic salts and oxides of rare earth elements like, europium, and uranium, which
phosphoresce naturally. These do not need any sample pre-treatment and the

49

Molecular Spectroscopic
Methods-II

phosphorescence can be measured directly by taking the compounds in a powder

holder of the solid sample accessory of the instrument. The second type of
compounds, on the other hand, are those which exhibit phosphorescence when
adsorbed onto certain supports like paper, cellulose, silica, etc. The phosphorescence
spectrum of salicylic acid adsorbed onto a paper from a solution containing 1 M
sodium hydroxide and 1 M sodium iodide is given in Fig. 6.4.

Fig. 6.4: The phosphorescence spectrum of salicylic acid adsorbed onto a paper from a
solution containing 1 M sodium hydroxide and 1 M sodium iodide

This technique has been extensively used for the analysis of pesticides, polycyclic
aromatic hydrocarbon (PAH), biphenyls, etc. Polycyclic aromatic hydrocarbons are
well known group of environmental pollutants, which have been found in combustion
products of synthetic fuels, cigarette smoke, etc. These are extremely hazardous, and
some of these are established to be carcinogenic in nature. Reliable and cost effective
monitoring of PAHs needs rapid screening of samples. The luminescent spectrometry
is ideally suited for the purpose due to its sensitivity and simplicity. The phenomenon
of quenching is also used for sensitized and quenched phosphorescence at room
temperature. It finds extensive applications in polymer chemistry research.

6.8.2 Applications of Phosphorescence Measurements

The majority of the applications of phosphorescence measurement have been in the
fields of drugs and pharmaceutical and in the analysis of pesticides. A number of
drugs like, Phenobarbital, cocaine, procaine, chlorpromazine, salicylic acid and a
number of sulphonamide drugs exhibit phosphorescence. The technique has also been
used in the determination of a number of drugs in biological samples like, urine and
blood. Phosphorescence measurements have also been used in the determination of air
and water pollutants and for the analysis of impurities in polycyclic aromatic
hydrocarbons and petroleum products.
Analysis of Elements by Phosphorescence Spectroscopy

A number of ionic compounds also exhibit intense room temperature

phosphorescence. Several metal ions like transition elements (Cu, Zn, Nb, Gd) as well
as s-block elements (Be) have been analysed by phosphorimetry. In addition, those
elements which cause fluorescence quenching (e.g, Fe, Cu, Co, Ni, Cr) can be
analysed by phosphorimetry, however, these need to be analysed as coordination
complexes. The complexing ligands used are - dketones like dibenzoylmethane
(DBM) benzoylacetone or quinoline derivatives like 8-hydroxyquinoline, etc. It is
possible to analyse metals at very low concentration e.g., (Cu 0.001mg) Be (0.0004
g) of the element can be analysed.

50

It is also possible to determine metals in the mixtures. A Beryllium complex win

HDBM as Be (DBM)2 is extracted in carbon tetrachloride and is separated from group
of transition and representative elements thereby avoiding the step of separations to
eliminate interferences. Amongst non metals, boron is efficiently analysed from
marine environment, so also Nb (V) was extracted in oxine at pH 9.4 with chloroform
as the solvent. Be when complexed with DGM and pyridine is measured at 527 nm.

Applications of
Fluorimetry and
Phosphorimetry

Table 6.6: Complexing ligands for the determination of metal ions and the
detection limit of the methods
Metals analysed

Complexing Ligand

Detection limit
(ppb or g)

Cu2+

Etioporphyrin- II

0.001mg

Zn2+

Etioporphyrin- II

1.0mg

Be2+

Dibenzoylmethane (DBM)

B.

Dibenzoylmethane (DBM)

0.1ppb

B.

Benzoylacetone

0.4ppb

Be2+

2-(2 hydroxyphenyl)benzoxazole

10ppb

Nb5+

8-Hydroxyquinoline

0.18ppb

Gd3+

Bis (8-hydroxy-2-quinolyl)methylamine

Be2+

Dibenzoylmethane + pyridine

0.0004g

The rare earths and uranyl elements phosphoresce and a number of them, particularly
europium and terbium, are used as phosphors in lamps and TV tubes. The
phosphorescence intensity of the rare earths increases tremendously when they are
covalently bound to certain molecules and this feature has been used in the analysis of
transferin in blood.

SAQ 5
What are the limitations of phosphorimetry over fluorimetry as an analytical method?
...
...
...
...

6.9

SUMMARY

Fluorescence and phosphorescence spectrometry find extensive applications pertaining

to the quantitative and qualitative analysis in diverse areas. The applications of
fluorimetry and phosphorimetry to the physical and life sciences have evolved rapidly
during the past decade. The increased interest in these appears to be due to advances in
time resolution, methods of data analysis, and improved instrumentation.
The fluorescence analysis methods can be broadly put under two categories as the
direct and indirect methods. In direct methods, we measure the natural fluorescence of

51

Molecular Spectroscopic
Methods-II

the analyte whereas in the indirect methods, we either suitably derivatise the analyte to
be determined to a fluorescent species or explore for a fluorescent species whose
fluorescence is quenched by the analyte.
The quantitative applications of fluorescence measurements are due to the relationship
between the fluorescence intensity and concentration of the analyte. However, a
number of factors like self quenching, radiant energy absorption, self absorption and
presence of interfering species pose problems which are to be suitably addressed
before undertaking the analysis.
Fluorimetric analysis is adequately meeting the growing need of highly sensitive and
selective probes for the detection of metal ions in environmental and biological
samples. The methods based on the phenomenon of chemiluminescence are being
effectively used for the determination of NO-NO2 and SO2 as atmospheric pollutants.
One can detect as small as 1 ppb of NO2 or 5 ppb of SO2 gas by these methods.
Similarly, the presence of trace amounts of metal pollutants in water samples can also
be analysed by fluorimetry.
In the field of biological samples, especially the clinical samples highly selective and
sensitive determinations are being accomplished by fluorimetry. To facilitate such
determinations spectrofluorimeters fabricated to suit the need of specific sample to be
analysed are available. Many applications in the field of clinical chemistry are based
on enzyme catalysed reactions. For example, a simple, specific and rapid test for the
concentration of glucose in the blood is based upon the enzyme glucose oxidase. The
fluorimetric determination of presence of ions in the biological samples is generally
based on specific complex formation between the ion and a suitable reagent. Several
important biochemical analysis are done by combining the chromatographic separation
and fluorimetry. In these chromatographic methods coupled with fluorimetry use a
suitable developing fluorophore. The HPLC separations at room temperature are
preferred.
Bioluminescence is phenomenon of luminescence occurring in living system, or
compound extracted from living systems. The oxidation of luminol in alkaline solution
is a well known example of bioluminescence. This reaction can be used for the
determination of ATP- an important molecule present in the cell. A large number of
applications of fluorescence spectroscopy involve the analysis of inorganic
compounds including metals, non-metals, minerals and alloys. These applications are
based on different kinds of reactions leading to generation of fluorescence. These
include formation of binary or ternary complexes, substitution reaction, redox
reactions or enzymatic reactions, etc. The vast range of applications in the area of
inorganic species can be put into three groups. The first group includes the inorganic
species that have an intrinsic fluorescence; the other two groups are of the species that
give fluorescence on reacting with inorganic and organic reagents, respectively.
Several minerals like calcite, fluorite, rubies and zircon on exposure to UV radiation
start emitting fluorescence in the absence of fluorescence quenchers.
The applications of phosphorescence are somewhat limited due to the lack of suitable
instrumentation and the requirement of making measurements at cryogenic
temperatures. However, with the introduction of new instrumental methods and the
advances made in room temperature phosphorescence, phosphorimetry is making
inroads in the domain of clinical chemistry and in the areas of forensic, environmental
and pharmaceutical sciences. In room temperature phosphorescence, the
phosphorescence spectra obtained from the analyte adsorbed onto solid supports like
filter paper, silica and other chromatographic supports. Several analytes are able to
give room-temperature phosphorescence (RTP) in organised media such as micelles
and cyclodextrin solutions. The presence of heavy atoms like, iodine, silver, leads, etc.

52

in the sample or in the solid matrix is found to improve the sensitivity of the technique
and helps in the analysis of complex mixtures by RTP.

Applications of
Fluorimetry and
Phosphorimetry

6.10 TERMINAL QUESTIONS

1.

What are the distinct advantages of using fluorescence spectroscopy methods

over UV-visible spectrophotometry?

2.

What do you understand by indirect method of fluorimetric determinations?

Illustrate with the help of an example.

3.

State the relationship between fluorescence intensity and the concentration of

the analyte. What precautions should be taken to exploit this relation in the
determination of very small concentrations of the analyte?

4.

What is chemiluminescence? Discuss its application in the determination of

NO-NO2 in a sample of polluted air.

5.

What is fluorescence quenching method of analytical determination of the

analyte? How is this method used for the analysis of fluoride from aquatic
environment?

6.

Which is the field wherein fluorescence spectroscopy has proved as the great
asset in quantitative chemical analysis?

7.

Enumerate various chemical reactions that may lead to the formation of

fluorinating species.

8.

What is the role of temperature in phosphorescence measurements?

6.11 ANSWERS
Self Assessment Questions
1.

The following factors act as problem areas in the course quantitative

determinations of the analyte using fluorescence spectrometry

Self quenching
Radiant energy absorption
Self absorption
Interfering species

2.

In the analysis of NO-NO2 by chemiluminescence ozone reacts with the NO in

the reactor to produce activated NO2 which in turn relaxes by emission of
photon.
NO + O
NO* + O

3.

Hydrogen peroxide is the product of oxidation reaction of glucose catalysed by

the enzyme glucose oxidase and is the molecule which is measured with the help
of a fluorophore named luminol as per the following reaction.

O
C
H2 O2

O
NH
NH

C
NH2

Luminol

C
base

O
O

catalyst

C
NH2

3-Aminophthalate*

C
NH2

3-Aminophthalate

53

Molecular Spectroscopic
Methods-II

Borate and fluoride are two ions which are extensively determined by
fluorescence measurements.
4.

In comparison to the fluorescence method, phosphorescence measurements find

very few applications. Some of the reasons for limited applications are as
follows:

Lack of suitable instrumentation,

The requirement to make measurements at cryogenic temperatures,

Longer phosphorescence life time leading to radiationless deactivation,

and

Quenching of phosphorescence by oxygen.

Terminal Questions
1.

Spectrophotometry as well as fluorimetry can be exploited for the quantitative

determinations of the analyte concentrations. However, fluorescence
spectrometry scores over spectrophotometry as in terms of:

Sensitivity

Selectivity

Availability of two wavelengths (excitation and emission)

2.

In one of the indirect analysis method of fluorimetry the analyte to be

determined is suitably derivatised to a fluorescent species. For example, a
nonfluorescent metal ion can be reacted with salicyaldehyde and the resulting
species is fluorescent.
H

C
O
H

M+

H+

3.

The fluorescence intensity of a sample is directly proportional to the

concentration of the analyte,
Pf c
In order to put this equation to use we need to eliminate all the causes that may
contribute to background signal.

4.

Chemiluminescence refers to the emission of radiation as a consequence of a

chemical reaction. In order to determine the amount of NO, the gas is passed
through the reactor in which it reacts with ozone to produce excited NO* as per
2

the reaction given below.

NO + O3
NO*2 + O2
The activated NO*2 then gives chemiluminescence broadband in the visible to
infrared range (600 2800 nm) and reverts back to a lower energy state.
5.

54

In this indirect method for fluorimetric determination of the analyte we look for
a fluorescent species whose fluorescence is quenched by the analyte. The

fluoride ions can be determined using the method based on quenching of the
fluorescence of Zr-alizarin complex. The complex is obtained at a pH 4.6 and
has the excitation and emission wavelengths as 470 and 520 nm, respectively.
The quenching of fluorescence is directly proportional to the concentration of
fluoride in water.
6.

Inorganic compounds like HCl, HBr react with metals like Tl, Sn, Pb, As, Sb, Bi
to generate fluorescence of sufficient intensity which can be measured at low
temperature. As regards organic ligands like Eosin (Ag, Pb), Morin (Al,Be,Mo),
Calcons (Cd,Mg), PAN( Co,Ni) can be quantitatively analysed by fluorimetric
measurements at optimum pH. They generally form binary and ternary
complexes and few of them show quenching (e.g., Mo).

7.

There are various kinds of the chemical reaction which produce fluorescence.
Important among them are binary or ternary complexation, substitution reaction,
redox reaction, kinetic method, and the direct extractive fluorimetric analysis.
Very low concentration of metals can be analysed by any of these reactions.

8.

The phosphorescence emission results from the transition from a triplet to a

singlet state. As the life time of a triplet state is long, the molecule in the excited
state gets enough of opportunities to collide with other molecules and lose
energy in nonradiative processes. Therefore, typically the phosphorescence
measurements are made at cryogenic temperatures. However, recent
developments leading to the possibility of getting reasonable phosphorescence
spectra at room temperature have opened newer areas to analytical application
of phosphorescence.

Applications of
Fluorimetry and
Phosphorimetry

55

Molecular Spectroscopic
Methods-II

56

SOME USEFUL BOOKS

1.

S.G. Sehulman (1985), Molecular Luminescence Spectroscopy Part I: Methods

and Applications. Wiley Interscience

2.

R.J. Hurtubise (1990), Phosphorimetry Theory, Instrumentation and

Application., VCH publication, New York

3.

J.A Rodley, J. Grant (1959), Fluorescence Analysis in UV light. Chapman and

Hall Co.

4.

S.G. Schulman (1977), Fluorescence and Phosphorescence Spectroscopy

Physicochemical Principles and Practices. Pergamon

5.

E.L Wehry(1976), Modern Fluorescence Spectroscopy, Vol.2. Plenum Press

6.

J.R Lakowicz (1991), Topics in Fluorenscence Spectroscopy, Vol.3. Plenum

Press

7.

D.A. Skoog, F.J. Holler, S.R. Crouch(2006), Principles of Instrumental

Analysis, 6th edition. Brooks Cole

8.

S.M. Khopkar (2008), Basic Concepts in Analytical Chemistry, 3rd edition. New
Age International Publishers

INDEX
Activation 8
Analysis of aluminum 36
Analysis of amino acids and proteins 38
Analysis of blood serum 40
Analysis of calcium ion 40
Analysis of creatinine phosphokinase 40
Analysis of elements by phosphorescence
spectroscopy 50
Analysis of fluoride 37
Analysis of gaseous pollutants 33
Analysis of water pollutants 36
Analysis of zinc 36
Applications of fluorescence and
phosphorescence 23
Applications of phosphorescence
measurements 50
Bioluminescence 41
Charge coupled device 20
Chemical reactions producing fluorescence
Binary complexation 43
Enzymatic reactions 43
Extractive fluorimetry
Kinetic reactions 44
Redox reactions 43
Substitution reactions 43
Ternary complexation 43

Chemiluminescence 6, 33, 34, 35, 41

Concentration dependence of fluorescence 31
Cuvette 15, 20
Deactivation 8
Determination of NO-NO2 33
Determination of SO2 34
Emission fluorescence spectrum 10
Excitation 8, 9
Excitation spectrum 11
Factors affecting fluorescence
Dissolved oxygen 14
pH 14
Solvent 15
Temperature 14

Factors affecting quantitative applications of

fluorimetry
Interfering species 32
Radiant energy absorption 32
Self absorption 32
Self quenching 32

Flow cell 20
Fluorescamine 38
Fluorescence
Fluorescence and mineral analysis 47
Fluorescence spectroscopy in medicine and
biology 37
Fluorescence quenching 15
Fluorescence spectrum 10, 11
Fluorescence with inorganic reagents 44
Fluorescence with organic reagents 45
Fluorescent and phosphorescent species 11
Fluorescent tag or label 13
Fluorimetry and environmental monitoring
fluorophore 33
Fluorimetric analysis of inorganic substances
42

Fluorimetric determination of blood

glucose 39
Fluorimetric method 35
Fluorescence analysis methods

Applications of
Fluorimetry and
Phosphorimetry

Direct analysis methods 29

Indirect analysis methods 29

Fluoroscence spectroscopy in quantitative

analysis 30
Franck-Condon principle 8
Inorganic substances showing
luminescence 44
Instrumentation for fluorescence
measurement
Detector 20
Read out Devices 20
Sampling 20
Sources 18
Wavelength selectors 19

Instrumentation for phosphorescence 21

Internal conversion 8
Intersystem crossing 9
Jablonski diagram 24, 26
Luciferin 42
Luciferyl adenylate 42
Luminescence 6, 12
Nonradiative deactivation 8
o-Phthalaldehyde 38
Phosphorescence 5, 6, 9
Phosphorimetric methods in chemical
analysis 48
Photoluminescence 6, 12
Photoluminescence and structure 12
Photomultiplier tubes 20
Quantum efficiency 12, 16, 31
Quantum yield 16
Quenching 15
Quenching constant 16
Radiative deactivation 8
Recording Procedure 22
Room temperature Phosphorescence 48
Self absorption 15
Self-quenching 15
Sensitised fluorescence 15
Singlet state 9
Spin-exchange 9
SternVolmer equation 15
Stokes shift 11, 24
Triplet (parallel)
Vibrational relaxation 8, 13
Wavelength Selectors 19
West Geake method 34
Xenon arc lamp 18

57