Spectrofluorimetry or

fluorimetry

HistoryThe term fluorescence comes from the mineral fluorspar (calcium fluoride) when Sir George G. Stokes observed in 1852 that fluorspar would give off visible light (fluoresce) when exposed to electromagnetic radiation in the ultraviolet wavelength.

Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon

LUMINESCENCEFLUORESCENCE

PHOSPHORESCENCESPECTROSCOPYCHEMILUMINESCENCE

LUMINESCENCE SPECTROSCOPY

Absorption first -

Followed by emission

in all directions , usually

at a lower frequency

LUMINESCENCE SPECTROSCOPY

• In favorable cases, luminescence methods are amongst some of the most sensitive and selective of analytical methods available.

• Detection Limits are as a general rule at ppm levels for absorption spectrophotometry and ppb levels for luminescence methods.

• When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence.

• Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off.

• The substances showing this phenomenon are known as flourescent substances.

• When light radiation is incident on certain substances they emit light continuously even after the incident light is cut off.

• This type of delayed fluorescence is called phosphorescence.

• Substances showing phosphorescence are phosphorescent substances.

• Chemiluminescence is based upon emission of light from an excited species formed as a result of a chemical reaction.

• Collectively, fluorescence and phosphorescence are known as photoluminescence

• Fluorimetry is the most commonly used luminescence method.

• Phosphorimetry usually requires at liquid nitrogen temperatures (77K).

• The terms fluorimetry and fluorometry are used interchangeably in the chemical literature.

Energy Level Diagram

s2

SINGLET STATES TRIPLET STATES

GroundState

s1 T

T

1

2

INTERSYSTEMCROSSING

VIBRATIONALRELAXATION

FLUORESCENCE PHOSPHORESCENCE

INTERNALCONVERSION CONVERSION

INTERNAL

• Following absorption of radiation, the molecule can lose the absorbed energy by several pathways.

• One competing process is vibrational relaxation which involves transfer of energy to neighbouring molecules which is very rapid in solution (10-13 sec).

• In the gas phase, molecules suffer fewer collisions and it is more common to see the emission of a photon equal in energy to that absorbed in a process known as resonance fluorescence.

• In solution, the molecule rapidly relaxes to the lowest vibrational energy level of the electronic state resulting due to internal conversion which shifts the molecule from S2 to an excited vibrational energy level in S1.

• Following internal conversion, the molecule loses further energy by vibrational relaxation. Because of internal conversion and vibrational relaxation, most molecules in solution will decay to the lowest vibrational energy level of the lowest singlet electronic state before any radiation is emitted.

• When the molecule has reached the lowest vibrational energy level of the lowest singlet electronic energy level then a number of events can take place.

• the molecule can lose energy by internal conversion without loss of a photon of radiation (least likely).

• the molecule can emit a photon of radiation equal in energy to the difference in energy between the singlet electronic level and the ground-state, this is termed fluorescence.

• the molecule can undergo intersystem crossing which involves an electron spin flip from the singlet state into a triplet state.

• Following this the molecule decays to the lowest vibrational energy level of the triplet state by vibrational relaxation.

• the molecule can then emit a photon of radiation equal to the energy difference between the lowest triplet energy level and the ground-state in a process known as phosphorescence.

Fluorescence & Phosphorescence

• In fluorescence, the lifetime of the molecule in the excited singlet state is 10-9 to 10-7 sec.

• In phosphorescence, the lifetime in the excited singlet state is 10-6 to 10 sec (because a transition from T1 to the ground state is spin forbidden).

the principle of spectrofluorometer

• It is an analytical device depends on the fluorescence phenomenon which is a short-lived type of photoluminescence created by electromagnetic excitation.

• That is, fluorescence is generated when a molecule transmits from its ground state So to one of several vibrational energy levels in the first excited electronic state, S1, or the second electronic excited state, S2, both of which are singlet states.

• Relaxation to the ground state from these excited states occurs by emission of energy through heat and/or photons.

• The difference between the excitation and emission wavelengths is called the Stokes shift.

• Stokes’ studies of fluorescent substances led to the formulation of Stokes’ Law, which states that the wavelength of fluorescent light is always greater than that of the exciting radiation. Thus, for any fluorescent molecule, the wavelength of emission is always longer than the wavelength of absorption.

Classification

• Based on the wavelength of emitted radiation when compared to absorbed radiation– Stokes fluorescence: wavelength of emitted

radiation is longer than absorbed radiation– Anti-stokes’s fluorescence: wavelength of emitted

radiation is shorter than absorbed radiation.– Resonance fluorescence: wavelength of emitted

radiation is equal to that of absorbed radiation.

FLUORESCENCE AND CHEMICAL STRUCTURE

Fluorescence is most commonly observed in compounds containing aromatic functional groups with low energy.

Most unsubstituted aromatic hydrocarbons show fluorescence - quantum efficiency increases with the no: of rings and degree of condensation.

CONTD…Simple heterocyclic do not exhibit

fluorescence.

Fusion of heterocyclic nucleus to benzene ring increases fluorescence.

Substitution on the benzene ring shifts wavelength of absorbance maxima and corresponding changes in fluorescence peaks

Fluorescence decreases with increasing atomic no: of the halogen.

Substitution of carboxylic acid or carboxylic group on aromatic ring inhibits fluorescence.

Fluorescence is favored in molecules with structural rigidity.

organic chelating agents complexed with metal ion increases fluorescence.

• Experimentally it is found that fluorescence is favoured in rigid molecules, eg., phenolphthalein and fluorescein are structurally similar as shown below. However, fluorescein shows a far greater fluorescence quantum efficiency because of its rigidity.

phenolphthalein

• It is thought that the extra rigidity imparted by the bridging oxygen group in Fluorescein reduces the rate of nonradiative relaxation so that emission by fluorescence has sufficient time to occur.

Fluorescein

Fluorescence Spectra

• Photoluminescence spectra are recorded by measuring the intensity of emitted radiation as a function of either the excitation wavelength or the emission wavelength.

• The excitation spectra is determined by measuring the emission intensity at a fixed wavelength , while varying the excitation wavelength. It is useful for selecting the best excitation wavelength for a quantitative or qualitative analysis.

• The emission spectra is determined by measuring the variation in emission intensity wavelength for a fixed excitation wavelength.

What is The fluorescence quantum yield (Φf)?

• It is the quantitative expression of the fluorescence efficiency, which is the fraction of excited molecules returning to the ground state by fluorescence.

• Quantum yields range from 1, when every molecule in an excited state undergoes fluorescence, to 0 when fluorescence does not occur.

• A molecule’s fluorescence quantum yield is influenced by external Variables such as:

• temperature• viscosity of solvent• pH• Increasing temperature generally decreases Φf

because more frequent collisions between the molecule and the solvent increases external conversion.

• Decreasing the solvent’s viscosity decreases Φf for similar reasons.

• For an analyte with acidic or basic functional groups, a change in pH may change the analyte’s structure and, therefore, its fluorescent properties.

What can specrofluorometer do?

• It has been used for the direct or indirect quantitative and qualitative analysis by measuring the fluorescent intensity F.

• It is relatively inexpensive and sensitive (the sensitivity of fluorescence is approximately 1,000 times greater than absorption spectrophotometric methods).

• fluorescent intensity F is dependent on both intrinsic properties of the compound (fluorescence quantum yield Φf), and on readily controlled experimental parameters including:

• intensity of the absorbed light I0

• molar absorption coefficient Ɛ• path length of the cell b• concentration of the fluorophor in solution c

• At low concentrations of fluorophore, the fluorescence intensity of a sample is essentially linearly proportional to concentration.

• However, as the concentration increases, a point is reached at which the intensity increase is progressively less linear, and the intensity eventually decreases as concentration increases further.

fluorescence intensity

concentrations

• The most common reason for this is Inner filter effect that, as the concentration of the sample increases, the light intensity experienced by some of the fluorescent molecules is lower than that experienced by others. When excitation intensity decreases, so does fluorescence emission intensity.

• It is generally necessary to use concentrations that result in absorbance values of 0.1 or lower to observe concentration dependent emission.

• As the concentration of molecules in a solution increases, probability increases that excited molecules will interact with each other and lose energy through processes other than fluorescent emission. Any process that reduces the probability of fluorescent emission is known as quenching.

• Other parameters that can cause quenching include:• presence of impurities• increased temperature• reduced viscosity of the solution media

Decrease in fluorescence intensity due to specific effects of constituents of the solution.

Due to concentration, ph, pressure of chemical substances, temperature, viscosity, etc.

Types of quenching Self quenching

Chemical quenching Static quenching Collision quenching

Fluorescence

Concentration of fluorescing species

Deviations at higher concentrations can be attributed to self-quenching or self-absorption.

Fluorescence

Concentration of fluorescing species

Calibration curve(Low con)

calibration curve(High con)

Here decrease in fluorescence intensity due to the factors like change in pH, presence of oxygen, halides &heavy metals.

pH- aniline at pH 5-13 gives fluorescence but at pH <5 &>13 it does not exhibit fluorescence.

halides like chloride,bromide,iodide & electron withdrawing groups like NO2,COOH etc. leads to quenching.

Heavy metals leads to quenching, because of collisions of triplet ground state.

This occurs due to complex formation. e.g.. caffeine reduces the fluorescence of

riboflavin by complex formation.

COLLISIONAL QUENCHING

It reduces fluorescence by collision. where no. of collisions increased hence quenching takes place.

Factors affecting fluorescence intensity• Conjugation: molecule must have conjugation ( π electron) so that uv/vis

radiation can be absorbed• Nature of substituent groups:

– e- donating groups like NH2, OH groups enhance fluorescence.– e- withdrawing groups like NO2, COOH reduce fluorescence.

• Fluorescent intensity is directly proportional to concentration.• Increase in viscosity leads to decreased collisions of molecules there by

increasing fluorescent intensity.• More rigid the structure of molecule, more the intensity of fluorescence.• Increase in temp leads to increased collisions b/w molecules decreasing

fluorescent intensity.• Presence of O2 decreases the fluorescence and so de-aerated solutions

must be used and compare result obtained from that of O2 containing solution.

INSTRUMENTATION

SOURCE OF LIGHT

FILTERS AND MONOCHROMATORS

SAMPLE CELLS

DETECTORS

Components of fluorimeters and spectrofluorimeters

Spectrophotometer V/S Spectroflorimeter

• The fluorescence is often viewed at 90° orientation (in order to minimise interference from radiation used to excite the fluorescence).

• Spectroflorimeter has two monochromators• As fluorescence is maximum between 25-30˚C, the sample holder

has the device to maintain the temperature

MERCURY ARC LAMP.XENON ARC LAMP.TUNGSTEN LAMP.TUNABLE DYE LASERS.

Sources of light

In fluorimeter 10 filter (absorb Vis. radiation and transmit UV radiation) and 20 filter (absorb UV radiation and transmit Vis. radiation) are present.

In spectrofluorometers, excitation monochromators (isolates only the radiation which is absorbed by the molecule) and emission monochromator (isolates only the radiation which is emitted by the molecule) are present.

Filters and Monochromators

Sample and sample holder

The majority of fluorescence assays are carried out in solution.

Cylindrical or rectangular cells fabricated of silica or glass used.

Path length is usually 10mm or 1cm.All the surfaces of the sample holder are

polished in fluorimetry.

Detectors PHOTOVOLTAIC CELL

PHOTO TUBE

PHOTOMULTIPLIER TUBES – Best and accurate.

Read out devices

The output from the detector is amplified and displayed on a readout device which may be a meter or digital display.

Microprocessor electronics provide outputs directly compatible with printer systems and computers, eliminating any possibility of operator error in transferring data.

SINGLE BEAM FLUORIMETER

DOUBLE BEAM FLUORIMETER

SPECTROFLUORIMETER(DOUBLE BEAM)

Source of light. The primary filter absorbs visible radiation and

transmits UV radiation. Emitted radiation measured at 90o by

secondary filter. Secondary filter absorbs UV radiation and

transmits visible radiation.Advantages • Simple in construction• Easy to use.• EconomicalDisadvantages• It is not possible to use reference solution & sample solution

at a time.• Rapid scanning to obtain Exitation & emission spectrum of the

compound is not possible.

Lamp

Primary filter

Secondary Filter

Photo Multiplier tube

I0

Ie

It

Similar to single beam instrument.Two incident beams from light source

pass through primary filters separately and fall on either sample or reference solution.

The emitted radiation from sample or reference pass separately through secondary filter.Advantages • Sample & reference solution can be analyzed

simultaneously.Disadvantages • Rapid scanning is not possible due to use of filters.

The primary filter in double beam fluorimeter is replaced by excitation monochromaters.

The secondary filter is replaced by emission monochromaters.

The incident beam is split into sample and reference beam using a beam splitter.

The detector is photomultiplier tube.

SPECTROFLUORIMETER

Advantages • Rapid scanning to get Exitation &

emission spectrum.• More sensitive and accuracy when

compared to filter fluorimeter.

• Advantages of fluorescence spectroscopy:SENSITIVITY : It is more sensitive as concentration is low as µg/ml or ng/ml.PRECISION : Upto 1 % can be achieved.SPECIFICITY : More specific than absorption method where absorption maxima may be same for two compounds.RANGE OF APPLICATION : Even non fluorescent compounds can also be converted to fluorescent compounds by chemical compounds.

• Disadvantages:Not useful for identification Not all compounds fluorescenceContamination can quench the fluorescence and hence give false/no results

Environmental Significance:• To detect environmental pollutants such as polycyclic

aromatic hydrocarbons:• pyrene• benzopyrene• organothiophosphorous pesticides• carbamate insecticides• Generally used to carry out qualitative as well as quantitative analysis for

a great aromatic compounds present in cigarette smoking, air pollutant concentrates & automobile exhausts

Geology:• Many types of calcite and amber will fluoresce under

shortwave UV. Rubies, emeralds, and the Diamond exhibit red fluorescence under short-wave UV light; diamonds also emit light under X ray radiation.

Applications of Spectrofluorometer:

Analytical chemistry:• to detect compounds from HPLC flow• TLC plates can be visualized if the compounds or a

coloring reagent is fluorescent• Plant pigments, steroids, proteins, naphthols etc can be

determined at low concentrationsBiochemistry:• used generally as a non-destructive way of tracking or

analysis of biological molecules (proteins)• Possible direct or indirect analysis aromatic amino

acids (phenylalanine- tyrosine-tryptophan)• Fingerprints can be visualized with fluorescent

compounds such as ninhydrin.

Medicine• Blood and other substances are sometimes detected by

fluorescent reagents, particularly where their location was not previously known.

• There has also been a report of its use in differentiating malignant, bashful skin tumors from benign.

Pharmacy:• Possible direct or indirect analysis drugs such as:• vitamins (vitamin A -vitamin B2 -vitamin B6 -vitamin B12 -vitamin E

-folic acid)• catecholamines (dopamine-norepinephrine)• Other drugs (quinine-salicylic acid–morphine-barbiturates –lysergic

acid diethylamide (LSD)) • to measure the amount of impurities present in the sample.

Fluorescent indicators • Intensity and colour of the fluorescence of many

substances depend upon the pH of solutions. These are called as fluorescent indicators and are generally used in acid base titrations.

• Eg: Eosin – pH 3.0-4.0 – colourless to green Fluorescien – pH 4.0-6.0 – colourless to green

Quinine sulphate: blue-violet. Acridine: green-violet

APPLICATIONS

EX 1. Determination of polyaromatic hydrocarbons

– Benzo[a]pyrene is a product of incomplete combustion and found in coal tar.

• Benzo[a]pyrene, is a 5-ring polycyclic aromatic hydrocarbon that is mutagenic and highly carcinogenic

• It is found in tobacco smoke and tar

• The epoxide of this molecule intercalates in DNA, covalently bonding to the guanine base nucleotide

Excitation and fluorescence spectra for benzo(a)pyrene in H2SO4. In the diagram the solid line is the excitation spectrum (the fluorescence signal is measured at 545 nm as the exciting wavelength is varied). The dashed line is the fluorescence spectrum (the exciting wavelength is fixed at 520 nm while the wavelength of collected fluorescence is varied).

Benzo(a)pyrene

EX 2. Fluorimetric Drug Analysis

• Many drugs possess high quantum efficiency for fluorescence. For example, quinine can be detected at levels below 1 ppb.

Quinine

• In addition to ethical drugs such as quinine, many drugs of abuse fluoresce directly. For example lysergic acid diethylamide (LSD) whose structure is:

LSD

• Because LSD is active in minute quantities, an extremely sensitive methods of analysis is required. Fluorimetricaly LSD is usually determined in urine from a sample of about 5mL in volume. The sample is made alkaline and the LSD is extracted into an organic phase consisting of n-heptane and amyl alcohol. This is a "clean-up" procedure that removes potential interferents and increases sensitivity. The LSD is then back-extracted into an acid solution and measured directly using and excitation wavelength of 335 nm and a fluorescence wavelength of 435 nm. The limit of detection is approximately 1 ppb.