2. spectrofluorimetry - aws · pdf file2. spectrofluorimetry both fluorescence and...

2. Spectrofluorimetry

Both fluorescence and phosphorescence are types of

photoluminescence (luminescence)

Luminescence: It is the process of reemission of previously absorbed light

When the molecules in the ground state absorb UV light, they are

transferred to the excited state, then, reemission of the previously absorbed

light takes place and the molecules return to the ground state where

fluorescence or phosphorescence takes place

Molecular Emission:

After the absorption of UV Visible light, the excited molecular

species are extremely short-lived and deactivation occurs due to:

a- Internal collision (internal conversion)

b- Cleavage of chemical bonds initiating photochemical reactions

c- Re-emission of light (luminescence)

d- Heat

e- Interaction between the solute and the solvent

Molecules on excitation normally posses’ higher vibrational energy

than they had in the ground state. This extra vibrational energy is lost (Fig.l )

by collision after which the molecules return to the ground electronic state with

the emission of light as fluorescence. Deactivation as fluorescence is a rapid

process occurring within 10-6 - 10-9 seconds of the excitation. Figure 1 shows

the energy transfer during the absorption, fluorescence and phosphorescence

of UV-Visible radiation. The lowest set of energy levels represents the ground

electronic level and its associated vibrational levels. The upper set of

Spectrofluorimetry

Instrumental analysis Dr. Hisham E Abdellatef

Page | 91

energy levels represents the first excited electronic state and its associated

vibrational sub-levels.

Photoluminescence should involve both photo-excitation and emission

processes

1- Photoexcitation process:

It may occur by absorption of one of the following forms of radiant

energy:

a- sunlight

b- visible radiation

c- UV

d- X-rays

2- The emission process:

It is the emission of radiant energy from an excited electronic state.

Photoluminescence is called fluorescence when the spin of the

excited electron does not change as the photoexcited species undergoes a

transition from the excited state to the ground state

Singlet and triplet state

Most of the organic compounds that fluoresce or phosphoresce are

aromatic. Some highly unsaturated aliphatic compounds with JI electronic

systems also yield luminescence. Each occupied orbital of a ground-state

molecule has a pair of electrons. The Pauli exclusion principle states that

two electrons in an orbital must have opposing spins and therefore, the net

spin for most ground state molecule is zero.

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In the excited molecules, which exhibit fluorescence, the spin of π electron

and that of π* electron, which together constitute a pi bond in the

chromophore system, are in opposite directions, i.e., they are anti-parallel,

and the molecules are in the singlet states. Some excited molecules

particularly at low temperature, may undergo a slow intersystem crossing

(the triplet excited state) in which the spin of π and π* electrons are

unpaired (parallel). Return from the triplet excited state to the singlet

ground state results in the emission of phosphorescence.

Intersystem crossing process is a slow process than fluorescence, and

consequently phosphorescence occurs after 10-8 seconds and may observed

even several minutes or hours after the source of excitation is removed. The

difference in the energy level (E) between the excited and unexcited

states during excitation (absorption), fluorescence and phosphorescence

are in the order: E (absorption)> E (fluorescence)> E (phosphorescence)

As the wavelengths corresponding to the E values are inversely

proportional to the wavelengths, the order of the max are:,(absorption) <

,(fluorescence) < (phosphorescence)

For example, the wavelengths of maximum excitation, fluorescence and

phosphorescence of anthracene are 255nm, 425nm, 680nm respectively. The

techniques of spectrofluorimetry and phosphorimetry measure the intensity

of the light emitted from a system that has absorbed radiant energy.

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Theory of fluorescence and phosphorescence:

The theory of luminescence is described by using a molecular-energy

interpretation. Fluorescence of organic molecules means emission of

radiant energy during a transition from the lowest excited singlet state S1

to the singlet ground state S0.

Phosphorescence of organic molecules means emission of radiant energy

during a transition from the lowest excited triplet state T1 to the singlet

ground state S0.

Figure 1: Partial energy diagram for a photoluminescent system

Spectrofluorimetry


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Theory of phosphorescence:

In phosphorescence, an intersystem crossing can take place readily

from S1 to one of the vibrational levels of T1 state that has very nearly the

same energy level (process III). This is followed by non radiative decay

(process IV) to the T1 level.

Intersystem crossing process involves a change in the spin of the

excited electron and thus a change in spin multiplicity.

The triplet state T1 is metastable, and molecules populating it have excess

energy. This energy can be lost by

a- Phosphorescence (process V)

b- Oxygen quenching: energy is transferred to molecular oxygen which

can easily undergo a transition since it has a triplet ground state. For

this reason oxygen must be excluded from the cuvet of

phosphorimeter.

c- By collision: collisions can be diminished by any process that makes a

sample rigid, so that the molecules are immobilized. This has been

done by increasing the viscosity of a suitable solvent by cooling it to a

point where a rigid glass is obtained. The solvent often used is "EPA" a

mixture of ethyl ether, isopropanol and ethanol in the ratio of 5: 5: 2

used at liquid nitrogen temperature (77K). This mixture does not,

crystallize.

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Differences between fluorescence and phosphorescence:

1- Phosphorescence may sometimes persist for many seconds after the

excitation source is removed.

2- Fluorescence emission is always at shorter wavelength than that of

phosphorescence.

3- Fluorescence is usually observed at room temperature in liquid

solution, while phosphorescence is observed in rigid medium at very

low temperature.

4- Fluorescence life time is usually in the range 10-7-10-9 sec, while

phosphorescence lifetime is usually in the range 10-4 -10 sec.

Half life time: It is the time required for half of the molecules to emit

photons and thus return to the ground states.

Effect of molecular structure on luminescence properties:

Fluorescence may be expected generally in:

1- Aromatic molecules that contain conjugated double bonds

2- Polycyclic aromatic compounds (with great number of π electrons)

3- Substituents strongly affect on the fluorescence; substituents such

as NH2, NHCH3, N(CH3)2, OH and OCH3 groups enhance the

fluorescence, while electron with drawing group such as NO2 Cl-,

Br-, I- and COOH groups decrease the fluorescence

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4- Rigid molecules are strongly fluorescent such as fluorescein and

eosin, while non rigid molecule such as phenolphthalein is not

fluorescent.

5- Formation of metal chelates promotes the fluorescence.

Phosphorescence may be expected generally in:

1- Aromatic hydrocarbons

2- Introduction of substituents such as NH2, SH, OH to aromatic

hydrocarbon enhance the phosphorescence and also aromatic nitro

compounds

3- Majority of aromatic aldehydes and ketons show phosphorescence.

Fluorescence Spectra:

Instruments that measure the intensity of fluorescence are called

fluorimeter. Those that measure the fluorescence intensity at variable

wavelengths of excitation and emission, and are able to produce fluorescence

spectra are called spectrofluorimeters

In the recording the fluorescence spectra, the limitations of light sources

and measuring devices assume real significance. These limitations are:

1- Variation of the intensity of available energy with .

2- Variation in the response of the detector to light of different

wavelengths.

In absorption spectrophotometry, both these factors are not

immediately evident, because comparison of blank and test solution is

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carried out under identical conditions, and the absorption spectrum

recorded is true (within the limitation of the instrument). The excitation and

fluorescence spectra, may, however, be grossly distorted version of the true

spectrum if the instrument is not specially adopted.

a) Excitation Spectra:

Before a compound can fluoresce, energy must be observed, and with

an ideal light source, of constant intensity at different wavelengths, the

most intense fluorescence is produced by radiation corresponding in

wavelength to that of the absorption peak of the substance. Therefore, if

the intensity of the fluorescence is plotted as a function of the wavelength

of the radiation used to excite the fluorescence, an activation or excitation

spectrum will, result. This will

be identical to the absorption

spectrum when corrected for

instrumental effect,

because the fluorescence

efficiency is greatly

independent of .

As the intensity of the

fluorescence is measured at a

particular wavelength, the

disadvantage of variation in sensitivity of the detector with the

Figure Fluorescence excitation and emission spectra for a solution of quinine

Spectrofluorimetry


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wavelength does not appear. However, in practice, the light source is not

ideal and the output from the monochromator used to supply exciting

radiation will vary according to wavelength. The detector will therefore

respond to variation in the intensity of the fluorescence caused by more or

less absorption of energy by the sample, and also by more or less

excitation energy available from the light source. A curve of intensity of

exciting light as a function of wavelength can be prepared for the light

source and may be used to correct the apparent excitation curve

obtained.

b) Emission Spectra (Fluorescence)

When a monochromator source of constant light intensity is used to

irradiate a sample, the fluorescence may be analysed in a monochromator

at constant slit width to give apparent emission spectrum. The true

spectrum is obtained by applying a correction for change in detector

sensitivity with wavelength and for changes due to fluorescence

monochromator i.e., half band width of emergent light and light losses.

Fluorescence emission spectra arise because of transition from the

first excited state and their shapes are therefore independent of the light

used to excite fluorescence. If the substance has an absorption band, the

emission spectrum often bears a mirror-image relationship to it when

plotted on a frequency scale, but if several bands occur, this relationship

may be highly distorted because of overlapping of absorption and

fluorescence bands.

Spectrofluorimetry


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Instrumentation:

When both the excitation and emission spectra are to be recorded,

two monochromator are essential, one for the light source (excitation

monochromator) and one for the fluorescence (emission

monochromator). The light source must provided a high level of UV and

Visible radiation and a compact high pressure Zenon arc lamp is used.

The production of ozone by the photochemical conversion of

atmospheric oxygen in the lamp compartment presents a toxic hazard

unless the ozone is thermally decomposed or removed by adsorption onto

charcoal. As many experiments will almost certainly entail the

measurement of very weak fluorescence. The detector must be a highly

sensitive photomultiplier tube of low dark current.

If the main interest lies in the fluorescence emission spectra, one

monochromator may be dispensed with a suitable light source and filter

Spectrofluorimetry


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used instead. The rather poor luminosity associated with the

monochromator even with a xenon arc lamp is replaced by the much more

intense light from a source such as a mercury vapour lamp, from which a

suitable activation beam is isolated by means of the filter. This

arrangement partially overcome, one of the difficulties inherent in

spectrofluorimetry, i.e., that so much of the available light is lost.

Advantages of spectrofluorimetry:

1-High sensitivity:

Substances that are reasonably fluorescent may be determined at

concentration up to 1000 times lower than those required for absorption

spectrophotometry. In spectrofluorimetric measurement, the

photomultiplier tube measures a single light intensity (relative to a zenon

light intensity) which may be amplified electronically many times without

introducing significant noise. In UV-Vis absorption spectroscopy, the

photomultiplier tube measure two intensities I0 and IT. at very low

absorbance, the small difference between I0 and IT approach the noise of

the signal and cannot be measured with satisfactory precision.

The high sensitivity offered by spectrofluorimetry may be of no

advantage if the sample contains a sufficient quantity of the analyte for

assay by absorption spectrophotometry, the latter being generally the

more precise technique. For example, the highly fluorescent substance

quinine sulphate may be assayed with good accuracy and precision in

Quinine sulphate tablets (300 mg) by measuring the absorbance at 348

Spectrofluorimetry


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nm of the filtered extract of the tablet powder in 0.1 M HCl. However, low

dosage drug formulations containing less than 1 mg per dose unit and

biological samples (blood, urine, etc....) containing low concentration of

the drugs, may require the high sensitivity of spectrofluorimetry, thus the

spectrofluorimetric method is of choice for the determination of many

hormones, alkaloids and vitamins in formulations and biological fluids.

2-Selectivity:

Two factors confirm on spectrofluorimetry a greater selectivity than

that given by UV-Visb. Absorption spectrophotometry. First, not all the

substances that absorb in the UV-Vis region fluoresce. In non fluorescent

molecules, absorbed energy is lost by alternative radiationless pathways,

principally by internal conversion. Molecules require in addition to a

chromophore, a degree of rigidity in their structure to reduce the

dissipation of the absorbed energy by internal conversion.

Substances that are fluorescent are characterized by their

wavelengths of maximum excitation and emission. Different fluorescent

species may show different wavelengths of maximum excitation and /or

emission. The facility to vary independently the wavelength of excitation

and the wavelength of fluorescence allows the analyst to select the

optimum combination of wavelength for the analyte and to reduce

interference from other fluorescing species in the sample,

Spectrofluorimetry


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Quantitative Aspects:

Many of the quantitative aspects of spectrofluorimetry may be

understood by reference to the fundamental equation for the intensity of

the fluorescence emitted. This equation may be derived from that of the

Beer-Lambert law:

A= LogI0/IT = abC

Or I0/IT =10abc

IT=I0 x l0-abc

but fluorescence (F) = (I0- IT)

where is the quantum yield of the fluorescence

At very low absorbance (< 0.02), the equation will be

F= 2.3 I0 abC

For a fixed set of instrumental (I0 and b) and sample (a and) parameters,

the fluorescence is proportional to the concentration.

F= K C where K = 2.3 I0 a b

Factors Affecting Fluorescence Intensity:

1- Concentration:

The previous equations show that the fluorescence intensity of a

substance is proportional to concentration only when the absorbance in a 1

cm cell is less than 0.02. With increasing the absorbance, introduce an

increasingly significant error (The inner filter effect) and cause negative

curvature in calibration graphs. If the concentration of the fluorescent

Spectrofluorimetry


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substance is so great that all incident radiation is absorbed, the equation

will be:

F = I0

That is the fluorescence is independent of concentration, and

proportional to the intensity of incident radiation only, a property that may

be utilized to determine the approximate emission characteristics of a light

source.

A further problem ensures if the emission and excitation spectra

overlap, which results in the reabsorption of fluorescence and a negative

dependence of fluorescence on the concentration. The variation over a

wide concentration range is shown in Fig. 3.

Fig. 3 Diagrammatic representation of the variation of fluorescence

intensity with concentration.

Region (a): Proportional relationship Region (b): Negative deviation from linearity. Region (c): Fluorescence independent of concentration Region (d): Reabsorption of fluorescence

Spectrofluorimetry


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Quantum yield of fluorescence ()

This is the ratio:

Since some absorbed energy is lost by radiationless pathways, the

quantum efficiency is less than 1.

Highly fluorescent substances take value near 1, which shows that most

of the absorbed energy is re-emitted as fluorescence. For example,

fluorescein in 0.1 M NaOH and quinine in 0.05 M H2SO4 have, values of

0.85 and 0.54 respectively at 23°C. Non-fluorescent substances have = 0.

2- Intensity of incident light (I0):

An increase in the intensity of light incident on the sample produces a

proportional increase in the fluorescence intensity. The intensity of

incident light depends on the intensity of light emitted from the lamp. The

excitation monochromator transmission properties (which for a particular

instrument are constant) and the excitation slit width. The intensity of

incident light and sensitivity of a fluorescence measured are increased by

increasing the width of excitation slit. However, wide slit settings

introduce problems due to photochemical decomposition or to spectral

overlap, with consequent reduction of selectivity. The choice of the

excitation slit-width is therefore a compromise between sensitivity,

selectivity and stability.

Spectrofluorimetry


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3- Pathlength (b):

The symbol for pathlength (b) in the previous equation does not

refer to the dimension of the sample cuvette but to the internal volume of

sample solution, when the fluorescence is both generated and detected.

The effective pathlength viewed by the detector depends on both the

excitation and emission slitwidths. Therefore, the use of microcuvettes

does not necessarily reduce the fluorescence. In fact, if inner filter

quenching or self-absorption is significant, the use of microcells may

reduce these interferences and actually increase the measured

fluorescence.

4- Adsorption:

The extreme sensitivity of the method requires very dilute solution,

10-100 times, weaker than those employed in absorption

spectrophotometry. Adsorption of the fluorescent substance on the

container walls may therefore presents serious problems and strong stock

solutions must be kept and diluted as required. Quinine is a typical

example of a substance which is adsorbed onto cell walls.

4-Oxygen:

The presence of oxygen may interfere in two ways:

a) By direct oxidation of the fluorescent substance to non-fluorescent

products.

b) By quenching of fluorescence. It is a useful precaution, therefore,

Spectrofluorimetry


Page | 106

to - check a de-aerated solution and compare the results obtained

with that from the oxygen-containing solution.

Anthracene is well known to be susceptible to the presence of oxygen.

5-pH:

It is to be expected that alteration of the pH of a solution will have a

significant effect on fluorescence if the absorption spectrum of the solute

is changed. Many phenols, for example, are fluorescent in both dissociated

and undissociated forms. Consequently, the fluorescence from a solution

of the phenol will show two peaks, one being due to the ionic form, acidic

solutions may be necessary to suppress the peak due to the ionic form.

6- Photodecomposition:

In absorption spectrophotometry, the intensity of the radiation

passing through solution is weak by photochemical standards, although

adequate for measurements; decomposition of the solute is therefore, not

very likely. Spectrofluorimetry, on the other hand, requires high intensity

illumination for irradiation, and the risk of photochemical change is

thereby increased. An error up to 20% could quite easily arise. It may be

possible in unfavourable cases to select radiation of a wavelength which is

not strongly absorbed so that the extent of photochemical change is

reduced, at the same time adequate sensitivity is retained.

Spectrofluorimetry


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7- Temperature and viscosity:

Variation in temperature and viscosity will cause variations in the

frequency of collision between molecules. Thus, an increase in the

temperature or the decrease in the viscosity is likely to decrease the

fluorescence by deactivation of the excited molecules by collision.

Similarly, many substances are not normally fluorescent at room temperature

are capable of emitting light when excited at a low temperature or when in a

viscous solvent or glassy matrix. The temperature coefficient of fluorescence

are typically-1%°C increase in the temperature.

8- Quenchers:

Quenching is the reduction of the fluorescence intensity by the

presence of substances in the sample other than the fluorescent

analyte(s). Absorption of the incident or emitted radiation quenches

the fluorescence by inner filter effect. Collisional quenchers reduce

the fluorescence by dissipating the absorbed energy or heat due to

collision with the quenching species. For example, quinine is highly

fluorescent in 0.05 M H2SO4 but not fluorescent in 0.1 M HC1 due to

collisional quenching by halide ions

Static Quenchers:

Form a chemical complex with the fluorescent substance and alter its

fluorescence characteristics. Certain xanthine derivatives e.g. caffeine,

reduce the fluorescence of riboflavine by static quenching. The application

of spectrofluorimetry for the study of binding properties (number of

Spectrofluorimetry


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binding sites, binding constant) of drugs and macromolecules e.g.

proteins, is based on the alternation of the fluorescence intensity of the

proteins or of the drug with the binding.

9- Scatter:

When the excitation and emission monochromators are at the same

wavelengths, scattered light of the same wavelength as the incident light

will be detected by the photomultiplier arising from colloidal particles in

the sample (Tyndall scatter) and from the molecules (Rayleigh scatter).

Even when the excitation and emission monochromators at set 20 nm or

vmore apart, a little Rayleigh- Tyndall scatter may be detected. Although it

is compensated by using a blank solution* it limits the sensitivity of the

measurements. Reduction of excitation and emission slit-widths to reduce

spectral overlap of excitation and emission spectra will reduce Rayleigh-

Tyndall scatter, at the expense of the sensitivity of the measurement.

Raman Scatter:

Arises from the conversion of some of the incident radiation into

vibrational and rotational energy by the solvent molecules. The resultant

scattered light is of lower energy and, consequently of longer wavelength.

Applications of spectrofluorimetry:

1- Compounds which are fluorescent are readily determined with simple

instruments as the solution for examination is normally obtained by

dissolution of the sample in a suitable solvent (Table 1)

Spectrofluorimetry


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Compound

PH

ax (nm)

Excitation Emission

Minimum

concentration

required (µg mL-1)

Adrenaline

1

295 335

0.1

Allyl morphine

1

285 355

0.1

Amylobarbitone

14

265 410

0.1

Chloroquin 11

335 400

0.05

Chlorpromazine

11

350 480

0.1

Cinchonidine

1

315 445

0.01

Cinchonine

1

320 420

0.01

Cyanocobalamine

7

275 305

0.003

Ergometrine

1

325 465

0.002

Folic acid

7

365 450

0.01

Menadione

280 320

0.07

Noradrenaline

1

285 325

0.006

Oxytetracycline

11

390 520

0.05

Reserpine

1

300 375

0.008

2- Single substances which are in themselves, non-fluorescent may be

determined as a result of chemical change (Table 2). This method is

useful for both inorganic and organic compounds, and many inorganic

compounds, form highly fluorescent complexes by combination with

organic reagents. The determination of selenium illustrates the increase in

the sensitivity which can be obtained with fluorimetry as as compared with

that for absorption. Thus 0.3 µgml-1 of selenium may be determined by

measurement of the absorbance of its complex with 3,3- diaminobenzidine,

but by using the fluorescence of the complex ,0.04 µg of selenium can be

Spectrofluorimetry


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measured. The sensitivity is further increased to 0.002 µg of selenium with

2,3 diaminonaphthalene as reagent.

e.g.:

1) Determination of primary amines, amino acids, peptides..etc. through:

Reaction with fluorescamine reagent

2) Determination of primary and secondary aliphatic amines through:

a- reaction with 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole ( NBD-CI ) give

yellow fluorescence

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

chloride)

Spectrofluorimetry


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Thiamine HCI in pharmaceutical preparations such as tablets and elixirs

and in food stuffs such as flour is relatively easily determined by oxidation to

highly fluorescent thiochrome. The product is soluble in 2-methyl-propan-1-ol

and hence is easily extracted from the reaction mixture for measurements

For mixture of two components, it may be possible to select the exciting radiation

of appropriate wavelengths, such that only one compound fluoresces at any

time. Even if there is not possible, measurements of the fluorescence at two

wavelengths may be sufficient to determine the composition of the mixture.

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