Unit 6 Applications of Fluorimetry and Phosphorimetry

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.

29

Molecular Spectroscopic Methods-II

H

H

C

C O H

O Salicyaldehyde

+

O

M+ O

+

H+

M

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, φ f , is defined by the following expression. φf =

=>

No. of photons emitted Intensity of fluorescen ce = No. of photons absorbed Intensity of absorption φf =

Pf Pa

… (6.1)

On rearranging this equation we get, Pf = ϕ f Pa As Pa = P0 − Pt , we may write, Pf = φf ( P0 − Pt ) = φf P0 (1 −

Pt

P0

)

… (6.2)

where, Po = incident radiant power, Pt = radiant power of emission spectra, Pf = fluorescence power, and φf = quantum yield of fluorescence. As Pt = P0 e −εbc => Pf = φt P0 [1‒ e −εbc ] 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 = φf P0 εbc (1−

εbc 2!

+

(εbc)2 ) 3!

… (6.3)

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

… (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

31

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 10‒12 moles can be measured. Keeping all other factors except concentration constant in Eq. 6.4, the final equation comes out to be: Pf α 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 β-amyloid peptides during the onset of Alzheimer’s 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 + hν (chemiluminescence) NO*2 M

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

33

Molecular Spectroscopic Methods-II

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 µg /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 + hν → SO*2 SO*2 → SO 2 + hν (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

C

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 λmax 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 + hν

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).

35

Molecular Spectroscopic Methods-II

Table 6.1: Atmospheric level of SO2 and NO2

Conc.

SO2 NO2

Concentration (ppb) Source vicinity Urban

Source 2 × 106

10

106

3

103

Rural

Remote

50- 100

5-50

1

20-500

5-50

3

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 µg 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 0.1µg / 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

N

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 µg/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.

37

Molecular Spectroscopic Methods-II

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 O

+

R-NH2

R

N

O OH

O Fluorescamine

Femto : 10‒15

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

+

N

R-NH2

R

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 β-D-glucose to form D-glucono-1,5-lactone and hydrogen peroxide.

β - 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 α -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

O

O

C

C

+ C NH2

O

Luminol

NH NH

base catalyst

C NH2

O

*

C

O O

O

3-Aminophthalate*

C NH2

O 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:

39

Molecular Spectroscopic Methods-II

NH2

NH2

O * O O

Intersystem crossing

+ N2

O

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.

O

Triplet dianion (T1) Excited State

NH2

O

* O

O

O

O

O Singlet dianion (S1) Excited State

+

hv

O Ground State dianion (S0)

The detection limit of glucose by this method is 50 µg /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.

λ excitation (nm)

λ emission (nm)

Sensitivity

1.

Brucine

H2O

7

305

500

Good

2.

Codeine

H2O

7

285

350

Poor

3.

Fluorene

Pentane

-

300

321

Good

4.

Hippuric acid

H2SO4

-

270

370

Good

5.

Indoles

H2O

7

315

350

Fair

6.

LSD

Acid

-

325

445

Good

7.

Morphine

H2O

7

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 + hυ 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

S

N

N

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, Schiff’s 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 1µg 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 µg/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) λ ex. = 260nm, λ 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 λ ex. = 350 nm and λ 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

λex

λem

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

F−

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 µg

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 µg

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 α (1→4) linkages. The three major cyclodextrins, α, β, and γ-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 ßß- 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 µ 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 µg)

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.

3

2

2

O C H2 O2

+

O NH NH

C NH2

O

Luminol

C base

O

*

C

O O

catalyst

O

C NH2

O

3-Aminophthalate*

O

C NH2

O

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

H

C

C O H

+

O

M+

O

O

+

H+

M

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.

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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

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Molecular Spectroscopic Methods-II

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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 Stern–Volmer equation 15 Stokes shift 11, 24 Triplet (parallel) Vibrational relaxation 8, 13 Wavelength Selectors 19 West Geake method 34 Xenon arc lamp 18

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