Pharmacy Dpt of QAU

1. ASSINGMENT NO#1
Fluorescence spectroscopy
Instrumentation
Submitted To : Dr. kifayat
Submitted By : Group B-5
Abdul Waris
Mahnoor Javed
Maria Nisar
Ramsha Umar
Shakeela Shakoor
Umama Abdul Qader
SubmitionDate : 26 October 2015
Department of Pharmacy
Qauid—i-Azam University , Islamabad

2. 2
Content
 Definition
 Introduction
 Principle
 Components
 Diagram
 Applications
 Limitation

3. 3
Fluorescence
spectroscopy
Definition:
Fluorescence spectroscopy is a type of electromagnetic spectroscopy which
analyzes fluorescence from a sample. Also known as fluorometry or
spectrofluorometry .
Introduction:
• Luminescence: emissionof photons from electronically excited states of
atoms, molecules, and ions.
• Fluorescence: Average lifetime from <10—10 to 10—7 sec from singlet states.
• Phosphorescence: Average lifetime from 10—5 to >10+3 sec from triplet
excited states.
• Fluorescence spectroscopy involves using a beam of light, usually ultraviolet
light, that excites the electrons in molecules of certain compounds and causes
them to emit light; typically, but not necessarily, visible light.
• A complementary technique is absorption spectroscopy.
Principle:
• Molecules have various states referred to as energy levels.
• Fluorescence spectroscopy is primarily concerned with electronic and vibrational
states.
• Generally, the species being examined has
a ground electronic state (a low energy
state) of interest, and an excited electronic
state of higher energy. Within each of these
electronic states are various vibrational
states.
• In fluorescence spectroscopy, the species is
first excited, by absorbing a photon, from its
ground electronic state to one of the various
Vibrationa
l state

4. 4
vibrational states in the excited electronic state.
• Collisions with other molecules cause the excited molecule to lose vibrational
energy until it reaches the lowest vibrational state of the excited electronic state.
• The molecule then drops down to one of the various vibrational levels of the
ground electronic state again, emitting a photon in the process.
• As molecules may drop down into any of several vibrational levels in the ground
state, the emitted photons will have different energies, and thus frequencies.
• Therefore, by analysing the different frequencies of light emitted in fluorescent
spectroscopy, along with their relative intensities, the structure of the different
vibrational levels can be determined.
ATOMIC FLUORESCENCE:"resonance fluorescence"
• For atomic species, the process is similar; however, since atomic species do not
have vibrational energy levels, the emitted photons are often at the same
wavelength as the incident radiation.
• This process of re-emitting the absorbed photon is "resonance fluorescence"
and while it is characteristic of atomic fluorescence, is seen in molecular
fluorescence as well.
Difference between fluorescence (emission) and
fluorescence excitationmeasurement :
• In a typical fluorescence (emission) measurement, the excitation wavelength is
fixed and the detection wavelength varies.
• While in a fluorescence excitation measurement the detection wavelength is fixed
and the excitation wavelength is varied across a region of interest.
Component:
 Light sources: Commonlyemployedsourcesinfluorescence spectrometryhave
spectral outputseitherasa continuumof energyoverawide range or as a seriesof discrete
lines.
An example of the firsttype isthe tungsten-halogenlampandof the latter,a mercurylamp.
Mercury lampsare the mostcommonlyemployedlinesourcesandhave the propertythattheir
spectral outputdependsuponthe pressure of the fillergas.The output froma low-pressure
mercurylampis concentratedinthe UV range,whereasthe mostcommonlyemployedlamps,of

5. 5
mediumandhighpressure have anoutputcoveringthe whole UV-visiblespectrum.
 Wavelength selection: The simplestfilterfluorimetersuse fixedfilterstoisolate
boththe excitedandemittedwavelengths.Toisolate one particularwavelengthfromasource
emittingaline spectrum,apairof cut-off filtersisall thatisrequired.Thesemaybe eitherglass
filtersorsolutionsincuvettes.The emissionfiltermustbe chosensothat the Rayleigh-Tyndall
scatteredlightisobscuredandthe lightemittedbythe sample transmitted.Toavoidhighblanks
it mayalso be necessarytofilteroutanyRaman scatter.
If monochromatorsare employed,itshould be possibletochange the slitwidthof boththe
excitationandemissionmonochromatorsindependently.Manyanalyseswill notrequirehigh
resolution(essentiallycorrespondingtohighselectivity) andgreatersensitivitywill be obtained
withwide slitwidths.Conversely,torecordthe fine structure inthe emissionof,for
example,,polyaromatichydrocarbonsorto excite selectivelyone compoundinthe presence of
another,narrowslitwidthswill be necessary,andsensitivitywill be sacrificed.
 Detectors: All commercial fluorescence instrumentsuse photomultipliertubesas
detectorsanda wide varietyof typesare available.The materialfromwhichthe photocathode
ismade determinesthe spectral range of the photomultiplierandgenerallytwotubesare
requiredtocoverthe complete UV-visible range.The S5type can be usedto detectfluorescence
out to approximately650 nm,but if itis necessarytomeasure emissionatlongerwavelengths,a
special redsensitive,S20,photomultipliershouldbe employed.
 Read-out devices: The outputfromthe detectorisamplifiedanddisplayedona
readoutdevice whichmaybe a meteror digital display.Itshouldbe possible tochange the
sensitivityof the amplifierinaseriesof discrete stepssothatsamplesof widely differing
concentrationcanbe compared.A continuoussensitivityadjustmentisalsouseful sothatthe
displaycanbe made to readdirectlyinconcentrationunits.
 Sample holders: The majorityof fluorescence assaysare carriedoutinsolution,the
final measurementbeingmade uponthe sample containedinacuvette orin a flowcell.Cuvettes
may be circular,square or rectangular(the latterbeinguncommon),andmustbe constructedof
a material thatwill transmitboththe incidentandemittedlight.Square cuvettesorcellswillbe
foundto be mostprecise since the parametersof pathlengthandparallelismare easierto
maintainduringmanufacture.However,roundcuvettesare suitableformanymore routine
applicationsandhave the advantage of beinglessexpensive.
The cuvette isplacednormal to the incidentbeam.The resultingfluorescence isgivenoff
equallyinall directions,andmaybe collectedfromeitherthe frontsurface of the cell,atright
anglestothe incidentbeam,orin-line withthe incidentbeam.

6. 6
Diagram:
Five essential components of fluorescence spectrometer
Application:
 Fluorescence spectroscopy is used in, among others, biochemical, medical, and
chemical research fields for analyzing organic compounds. There has also been
a report of its use in differentiating malignant skin tumors from benign.
 Atomic Fluorescence Spectroscopy (AFS) techniques are useful in other kinds of
analysis/measurement of a compound present in air or water, or other media,
such as CVAFS which is used for heavy metals detection, such as mercury.
 Fluorescence can also be used to redirect photons.
 Additionally, Fluorescence spectroscopy can be adapted to the microscopic level
using microfluorimetry
 In analytical chemistry, fluorescence detectors are used with HPLC

7. 7
Limitation:
 not useful for identification
 not all compounds fluorescence
 contamination can quench the fluorescence and hence give false results
 fluorescence is more sensitive to fluorophore environment due to increase
time , molecule stays in excited state
 substances which display fluorescence should have :
 rigid structures
 delocalised electrons
 intense UV absorption bands
 short excited state lifetimes
 good overlap between electron orbitals of ground and first excited states.

8. 8
References
 https://en.wikipedia.org/wiki/Fluorescence_spectroscopy
 http://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Fluor
escence%20Spectroscopy.pdf
 http://www2.warwick.ac.uk/services/ris/business/analyticalguide
/fluorescence/
 http://www.horiba.com/scientific/products/fluorescence-
spectroscopy/?gclid=COOPp4v22sgCFYEojgodBHMLeQ
 http://eu.wiley.com/WileyCDA/WileyTitle/productCd-
1405138912.html
 http://pubs.acs.org/doi/abs/10.1021/ac00169a713?journalCode=
ancham