FLUOROMETRY

BASIC CONCEPTS, INSTRUMENTATION AND

APPLICATIONS By

Dr. Basil, B – MBBS (Nigeria),Department of Chemical Pathology/Metabolic Medicine,

Benue State University Teaching Hospital, Makurdi.September 2014.

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INTRODUCTION:• 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.

FLUORESCENCE: occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength

• An atom or molecule that fluoresces is termed a Fluorophore

• Fluorometry is defined as the measurement of the emitted fluorescence light

• Fluorometric analysis is a very sensitive and widely used method of quantitative analysis in the chemical and biological sciences

• Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off 2

PHOSPHOFLUORESCENCE: This occurs when light radiation is incident on certain substances and they emit light continuously even after the incident light is cut off.

• Substances showing phosphorescence are phosphorescent substances.

Fluorescence and Phosphoflourescence:

• A molecular electronic state in which all of the electrons are paired are called singlet state.

• In a singlet state molecules are diamagnetic.

• Most of the molecules in their ground state are paired.

• When such a molecule absorbs uv/visible radiation, one or more of the paired electron raised to an excited singlet state /excited triplet state.

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• The electron spins in the excited state achieved by absorption of radiation may either be parallel or antiparallel. Accordingly, this may be a triplet (parallel) or a singlet (antiparallel) state.

Singlet and Triplet States:

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BASIC CONCEPTS: Jablonski Diagram

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• Each molecule contains a series of closely spaced energy levels

• Absorption of a quantum of light energy by a molecule causes the transition of an electron from the singlet ground state to one of the number of possible vibrational levels of its first singlet state

• Once the molecule is in an excited state, it returns to its original state in several ways. These are:– Radiation-less vibrational equilibration

– The fluorescence process from the excited singlet state

– Quenching of the excited singlet state

– Radiation-less crossover to a triplet state

– Quenching of the first triplet state, and

– The phosphorescence process of light emission from the triplet state. 6

• The vibrational equilibrium before fluorescence results in some loss of the excitation energy,

• thus, the emitted fluorescence is of less energy and longer wavelength than the excitation light

• The difference btw the max wavelength of the excitation light and the max wavelength of the emitted fluorescence lights is a constant – stokes shift

• This constant is a measure of energy lost during the lifetime of the excited state (RVD) before returning to the ground singlet level (fluorescence emission).

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Time Relationship of Fluorescence Emission:

• There is a considerable time delay btw the (1) absorption of light energy, (2) return to the lowest excited state, (3) emission of fluorescence light.

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• The time required for the emitted light to reach 1/e of its initial intensity , where e is the Naperian base 2.718, is called the average lifetime of the excited state of the molecule, or the fluorescence decay time

• The advantage of time-resolved fluorometer (time delay btw absorption of quanta of light and fluorescence) is the elimination of background light scattering as a result of Rayleigh and Raman signals and Short-lived fluorescence background

• Time-resolved fluorometry depending on how the fluorescence emission response is measured is categorized as:– Pulse flurometry: sample illuminated with as intense brief pulse

of light and the intensity of emission measured as a function of time with a fast detector

– Phase fluorometry: continuous-wave laser illuminates the sample, and the emission monitored for impulse and freqresponse.

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Relationship of concentration and fluorescence intensity:• Fluorescence intensity is directly proportional to the

concentration of the fluorophore and the excitation intensity

F = ФIo abcwhere F = relative intensity

Ф = fluorescence efficiency (i.e., the ration btw quanta of light emitted and quanta of light absorbed)

Io = initial excitation intensitya = molar absorptivityb = volume element defined by geometry of

the excitation and emission slitsc = the concentration in mol/L

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• This relationship holds only for dilute solutions, where absorbance is less than 2% of the exciting radiation. Higher than 2%, the fluorescence intensity becomes nonlinear – inner filter effect.

• The magnitude of fluorescence intensity of a fluorophore is determined by:– Its concentration

– The path length

– The intensity of the light source

• Fluorescence measurements are 100 – 1000 times more sensitive than absorbance measurement due to:– More intense light source

– Digital filtering techniques

– Sensitive emission photometers

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• Fluorescence measurements are expressed in “relative” intensity units because the intensity measured is not an absolute quantity and its magnitude is defined by the (instrument-related varriables):– Instrument slit width– Detector sensitivity– Monochromator efficiency– Excitation intensity

Fluorescence Polarization:• This is defined by:

P = (Iv – Ih)/(Iv + Ih)where Iv = intensity of emitted fluorescence light in vertical

planeIh = intensity of emitted fluorescence light in horizontal

plane

• Fluorescent polarization is measured by placing a mechanically or electrically driven polarizer btw the sample cuvet and detector

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• In normal instrumentation mode, the sample is excited with polarized light to obtain maximum sensitivity.

• The polarizer analyser is placed to measure the intensity of the emitted fluorescence both in vertical and horizontal planes before calculating for P.

• Fluorescence polarization is used to quantitate analytes by use of the change in fluorescence depolarization following immunological reactions

• This is done by adding a known quantity of fluorescent-labelled analyte molecule to a reaction solution containing an antibody specific to the analyte.

• The labeled analyte binds to the antibody resulting in a change in its rotational relaxation time and fluorescence polarization.

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QUENCHING:• Decrease in fluorescence intensity due to specific

effects of constituents of the solution e.g., concentration, ph, pressure of chemical substances, temperature, viscosity, etc.

• Types of quenching include:

– Self quenching

– Chemical quenching

– Static quenching

– Collision quenching

Collisional Quenching:

• It reduces fluorescence by collision. where no. of collisions increased hence quenching takes place.15

Self Quenching/ Concentration Quenching:

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Chemical Quenching:• 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.

Static Quenching:• This occurs due to complex formation e.g., caffeine

reduces the fluorescence of riboflavin by complex formation.

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PHOTODECOMPOSITION:

• Excitation of a weakly fluorescing or dilute solutions with intense light sources will cause photochemical decomposition of the analyte (Photobleaching). This is minimized by:

– Use of the longest feasible wavelength for excitation that does not introduce light-scattering effects.

– Measure the fluorescence immediately after excitation to decrease the duration of excitation

– Protect unstable solutions from ambient light by storing them in dark bottles

– Remove dissolved oxygen from the solution

• Decomposition introduces non-linear response curves and loss of the majority of the sample fluorescence.

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Factors affecting Fluorescence and PhosphorescenceTemperature/Viscosity:• The change in temperature causes the viscosity of the

medium to change which in turn changes the number of collisions of the molecules of the fluorophore with solvent molecules.

• A rise in temperature is almost always accompanied by a decrease in fluorescence.

• Increase in viscosity increases fluorescent intensity• The increase in the number of collisions between

molecules in turn increases the probability for deactivation by internal conversion and vibrational relaxation (Collisional Quenching).

• Temperature of the reaction must be regulated to within +/- 0.1’C 19

pH:

• Relatively small changes in pH can sometimes causesubstantial changes in the fluorescence intensity and spectralcharacteristics of fluorescence.

– For example, serotonin shows a shift in fluorescenceemission maximum from 330 nm at neutral pH to 550 nm instrong acid without any change in the absorption spectrum.

• In the molecules containing acidic or basic functional groups,the changes in pH of the medium change the degree ofionisation of the functional groups. This in turn may affect theextent of conjugation or the aromaticity of the molecule whichaffects its fluorescence.

– For example, aniline shows fluorescence while in acidsolution it does not show fluorescence due to the formationof anilinium ion.

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• Therefore, pH control is essential while working with such molecules and suitable buffers should be employed for the purpose.

Dissolved Oxygen:• The paramagnetic substances like dissolved oxygen

and many transition metals with unpaired electronsdramatically decrease fluorescence and causeinterference in fluorimetric determinations.

• The paramagnetic nature of molecular oxygenpromotes intersystem crossing from singlet to tripletstates in other molecules.

• The longer lifetimes of the triplet states increases theopportunity for radiationless deactivation to occur.

• Presence of dissolved oxygen influences phospho-rescence too and causes a large decrease in the phosphorescence intensity.

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• It is due to the fact that oxygen at the ground state gets the energy from an electron in the triplet state and gets excited.

• This is actually the oxygen emission and not the phosphorescence. Therefore, it is advisable to make phosphorescence measurement in the absence of dissolved oxygen.

Solvent:

• The changes in the “polarity” or hydrogen bonding ability of the solvent may also significantly affect the fluorescent behaviour of the analyte.

• The difference in the effect of solvent on the fluorescence is attributed to the difference in their ability to stabilise the ground and excited states of the fluorescent molecule. 22

• Besides solvent polarity, solvent viscosity and solvents with heavy atoms also affect fluorescence and phosphorescence.

• Increased viscosity increases fluorescence as the deactivation due to collisions is lowered.

• A higher fluorescence is observed when the solvents do not contain heavy atoms while phosphorescence increases due to the presence of heavy atoms in the solvent.

• Some solvents e.g Ethanol, also cause appreciable fluorescence.

• Other sample matrix, e.g protiens and bilirubin are more serious contributors to unwanted fluorescence

Adsorption:• Extreme sensitiveness of the method requires very dilute

solution. Adsorption of the fluorescent substances on the container wall create serious problems. Hence strong solutions must be diluted.

• Certain quartz glass and Plastic materials that contain ultraviolet adsorbers will fluoresce 23

Concentration Effects:

Concentration is proportional to the emitted light energy absorbed . At the maximum concentration, fluorescence peaks and may decrease thereafter.

• Concentration quenching

• Inner Filter Effect: refers to the deviation from a linear relationship btw concentration and fluorescence as the absorbance of the solution increases above 2% of the exciting light

– Thus, as the concentration increases, the absorbance of the excitation intensity increases, and the loss of the light as it travels through the cuvet increases.

Light effects:

• Type of light: Monochromatic light is essential for the excitation of fluorescence because the intensity will vary with wavelength.

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• Length of time of exposure: The number of molecules excited increase with the time of exposure hence decrease in fluorescence due to Photodecomposition

• Intensity of Incident light: Increase in intensity of light incident on sample increases fluorescence intensity. The intensity of light depends upon:– Light emitted from the lamp.– Excitation monochromaters.– Excitation slit width

• Light scattering:– Rayleigh scattering: Diffusion of radiation in the course

of its passage through a medium containing particles the size of which is small compared with the wavelength of the radiation• occurs with no change of wavelength

– Raman scattering occurs with lengthening of wavelength.• It is a property of the solvent and difficult to eliminate at low

fluorophore concentration25

Nature of Substituent:• Electron donating group enhances fluorescence –

.g.NH2,OH etc.• Electron withdrawing groups decrease or destroy

fluorescence. e.g.COOH,NO2, N=N etc.• High atomic no: atom introduced into the electron

system decreases fluorescence.

Path length:• The effective path length depends on both the

excitation and emission slit width.• Use of microcuvette does not reduce the

fluorescence.• Use of microcell may reduce interferences and

increases the measured fluorescence26

INSTRUMENTATION:

THE FLUOROMETER

Single-beam and Double-beam Instrument Designs

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Basic Components:• Basic components of fluorometers and Spectrofluorometers

include: – an excitation source, an excitation monochromator, a cuvet, an

emission monochromator, a detector.

• The optical geometry for fluorescence measurement is very important:– most commercial fluorometers use the right angle detector

approach because it minimizes the background signal that limits analytical detection.

– The end-on approach allows the adaptation of a fluorescence detector to existing 180 degrees absorption instrument.

– The front surface approach has a comparable limit of detection to right angle detectors and it minimizes the inner filter effect, but is more susceptible to background light scatter.

• To accommodate these different geometries, the sample cell is oriented at different angles in relation to the excitation source and the detector.

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Basic Components of Fluorometers:

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Geometrical arrangements of Fluorometres:

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• If the emission slit is located near the front edge of the sample cell, the inner filter effect is minimized.

• If the emission slit width is increased, the detector will be more sensitive, but specificity may decrease.

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Light Source:• Mecury Arc Lamp:

– Produce intense line spectrum above 350nm.– High pressure lamps give lines at 366,405, 436,

546,577,691,734nm.– Low pressure lamps give additional radiation at 254nm.

• Xenon Arc Lamp:– Intense radiation by passage of current through an

atmosphere of xenon.– Spectrum is continuous over the range between over 250-

600nm,peak intensity about 470nm• Tungsten Lamp:

– Intensity of the lamp is low.– If excitation is done in the visible region this lamp is used.– It does not offer UV radiation

• Turnable Dye Lasers:– Pulsed nitrogen laser as the primary source– Radiation in the range between 360 and 650 nm is

produced. 32

• Excitation monochromators:- isolates only the radiation which is absorbed by the molecule.

• Emission monochromaters:- isolates only the radiation emitted by the molecule.

• Primary filter:- absorbs visible light & transmits UV light.

• Secondary filter:- absorbs UV radiations & transmits visible light.

Cuvets:• 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.

Monochromators and Filters:

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Detectors:• These include:

– Photovoltaic Cell– Phototube– Photomultiplier tube: - best and accurate

• Photomultiplier Tube:– Multiplication of photo electrons by secondary

emission of radiation.– A photo cathode and series of dynodes are used.– Each cathode is maintained at 75-100v higher than the

preceding one.– Over all amplification of 106 is obtained.

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Single-Beam Fluorometers:• Tungsten lamp as 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 and 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.

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Double-Beam Fluorometer:• Two incident beams of 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 and Reference solution can be analysedsimultaneously

Disadvantage:

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

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APPLICATIONS:• Determination of Vitamin B1 and B2

• Liquid Chromatography:– It is an important method of determining compounds as

they appear at the end of chromatogram or capillary electrophoresis column

• Organic Analysis:– Qualitative and quantitative analysis of organic aromatic

compounds present in cigarette smoke, air pollutants, automobile exhausts etc.

• Fluorescent indicators:– Mainly used in Acid-Base titration e.g:

• Eosin: colourless – green

• Fluorescein: colourless – green

• Quinine sulphate: blue – violet

• Acridine: green – violet. 37

• Fluorometric reagent:

– Aromatic structure with two or more donor functional groups

• Pharmaceutical Analysis:

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• Other Applications include:

– Determination of inorganic substances: like Aluminum, boron, cadmuim with 2-(2OH-phenyl)benzoxazole in the presence of tartarate, ruthenuim

– Determination of Uranuim salts in Nuclear research

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REFERENCES:• Teitz Textbook of Clinical Chemistry and Molecular

diagnosis (5th Edition)

• Dr.B.K.Sharma, Instrumental methods of chemical analysis.

• Gurdeep R Chatwal, Instrumental methods of chemical analysis

• http://en.wikipedia.org/wiki/Fluorescence

• http://images.google.co.in/imghp?oe=UTF-8&hl=en&tab=wi&q=fluorescence

• http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm

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