1 instrumental analysis fundamentals of spectroscopy
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Instrumental Analysis Instrumental Analysis
Fundamentals of Fundamentals of SpectroscopySpectroscopy
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(Absorption) SpectrophotometryGeneral Stuff:
•Qualitative: Spectrum (a plot of A vs. ) ischaracteristic of a specific species•Quantitative: Absorbance at a particular can berelated to the amount of absorbing speciesDefinitions and units.monochromatic wavelength (cm)Po.incident radiant power (erg cm -2 s -1 )P .transmitted radiant power (erg cm -2 s -1 )b .absorption pathlength(cm)
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Qualitative analysis: The spectrum
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Molecular and Atomic SpectrometryMolecular and Atomic Spectrometry
Spectrometry is the study of Spectrometry is the study of electromagnetic radiation (EMR) and its electromagnetic radiation (EMR) and its applicationsapplications
To begin to understand the theory and To begin to understand the theory and instrumental application of spectrometry instrumental application of spectrometry requires an understanding of the requires an understanding of the interaction of EMR (i.e. light) with matterinteraction of EMR (i.e. light) with matter
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QuestionsQuestions
What is nature of light?What is nature of light?
Are their different types of light?Are their different types of light?
–How are they the same?How are they the same?
–How are they different?How are they different?
How does light propagate?How does light propagate?
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What is Light?What is Light?
Light is a form of energyLight is a form of energy
Light travels through space at extremely Light travels through space at extremely high velocities high velocities – The speed of light (c) ~ 3 x 10The speed of light (c) ~ 3 x 101010 cm/sec or cm/sec or
186,000 miles per second186,000 miles per second
Light is classified as electromagnetic Light is classified as electromagnetic radiation (EMR)radiation (EMR)
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Characteristics of LightCharacteristics of Light
Light behaves like a Light behaves like a wavewave..– That is, it can be modeled or characterized That is, it can be modeled or characterized
with wave like properties.with wave like properties.
Light also behaves like a Light also behaves like a particleparticle..– The photon and photoelectric effect.The photon and photoelectric effect.
Today, we envision light as a self-Today, we envision light as a self-contained packet of energy, a contained packet of energy, a photonphoton, , which has both wave and particle like which has both wave and particle like properties.properties.
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The Electromagnetic SpectrumThe Electromagnetic Spectrum
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The Electromagnetic SpectrumThe Electromagnetic Spectrum
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The EMR The EMR SpectrumSpectrum
Different portions of Different portions of the EMR spectrum the EMR spectrum and different types of and different types of spectroscopy involve spectroscopy involve different parts different parts (quantum states) of (quantum states) of the atomthe atom
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EMR Wave CharacteristicsEMR Wave CharacteristicsWavelength (Wavelength ()) is the distance from one wave crest to is the distance from one wave crest to the next.the next.AmplitudeAmplitude is the vertical distance from the midline of a is the vertical distance from the midline of a wave to the peak or trough.wave to the peak or trough.FrequencyFrequency ((vv)) is the number of waves that pass through is the number of waves that pass through a particular point in 1 second (Hz = 1 cycle/s)a particular point in 1 second (Hz = 1 cycle/s)
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EMR Wave CharacteristicsEMR Wave Characteristics
The frequency of a wave is dictated (or The frequency of a wave is dictated (or fixedfixed) by ) by its source, it doesn’t change as the wave passes its source, it doesn’t change as the wave passes through different mediums. through different mediums. The The speedspeed of a wave ( of a wave (uu), however, can change ), however, can change as the medium through which it travels changesas the medium through which it travels changes
uumediummedium == v = c/nv = c/n
Where n = refractive indexWhere n = refractive indexnnvacuumvacuum = 1 = 1
nnairair = 1.0003 (v = 1.0003 (vairair = 0.9997c) = 0.9997c)
nnglassglass ~1.5 (v ~1.5 (vgasgas ~ 0.67c) ~ 0.67c)
Since Since vv is fixed, as is fixed, as decreasesdecreases, , u u must also must also decreasedecrease
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Wave Properties of Wave Properties of Electromagnetic RadiationElectromagnetic Radiation
EMR has both electric (E) and magnetic EMR has both electric (E) and magnetic (H) components that propagate at right (H) components that propagate at right angles to each other.angles to each other.
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Particle Properties of EMRParticle Properties of EMR
TheThe energy energy of a photon depends on its of a photon depends on its
frequency (frequency (vv))
EEphotonphoton = = hhvv
hh = Planck’s constant= Planck’s constant
hh = 6.63 x 10 = 6.63 x 10-27-27 erg sec or 6.63 x 10 erg sec or 6.63 x 10-34-34 Js Js
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Relationship between Wave and Relationship between Wave and Particle Properties of EMRParticle Properties of EMR
EEphotonphoton = = hv ; uhv ; umediummedium == v = c/nv = c/n
With these two relationships, if you know one of With these two relationships, if you know one of the following, you can calculate the other twothe following, you can calculate the other two– Energy of photonEnergy of photon– Wavelength of lightWavelength of light– Frequency of lightFrequency of light
EEphotonphoton = = n
hc
EEphotonphoton = = hv ; uhv ; umediummedium == v = c/nv = c/n
With these two relationships, if you know one of With these two relationships, if you know one of the following, you can calculate the other twothe following, you can calculate the other two– Energy of photonEnergy of photon– Wavelength of lightWavelength of light– Frequency of lightFrequency of light
EEphotonphoton = = n
hc
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Relationship between Wave and Relationship between Wave and Particle Properties of EMRParticle Properties of EMR
Example: What is the energy of a 500 nm Example: What is the energy of a 500 nm photon?photon?
= c/= c/ = (3 x 10 = (3 x 1088 m s m s-1-1)/(5.0 x 10)/(5.0 x 10-7-7 m) m) = 6 x 10= 6 x 101414 s s-1-1
E = hE = h =(6.626 x 10 =(6.626 x 10-34-34 J J••s)(6 x 10s)(6 x 101414 ss-1-1) = 4 x 10) = 4 x 10-19-19 J J
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How Light Interacts with Matter.How Light Interacts with Matter.
Atoms are the basic Atoms are the basic blocks of matter.blocks of matter.
They consist of heavy They consist of heavy particles (called protons particles (called protons and neutrons) in the and neutrons) in the nucleus, surrounded by nucleus, surrounded by lighter particles called lighter particles called electronselectrons
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How Light Interacts with Matter.How Light Interacts with Matter.
An electron will interact with a photon.An electron will interact with a photon.
An electron that An electron that absorbsabsorbs a photon will a photon will gain gain energy.energy.
An electron that An electron that losesloses energy must energy must emitemit a a photon.photon.
The total energy (electron plus photon) The total energy (electron plus photon) remains constant during this process.remains constant during this process.
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Characteristics of AbsorptionCharacteristics of Absorption
AbsorptionAbsorption is defined as the process by is defined as the process by which EMR is transferred, in the form of which EMR is transferred, in the form of energy, to the medium (s, l, or g) through energy, to the medium (s, l, or g) through which it is travelingwhich it is traveling
Involves discrete energy transfersInvolves discrete energy transfers
Frequency and wavelength selectiveFrequency and wavelength selective
– EEphotonphoton = = hv = c/hv = c/
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Characteristics of AbsorptionCharacteristics of Absorption
Involves transitions from ground state Involves transitions from ground state energy levels to “excited” statesenergy levels to “excited” states– The reverse process is called The reverse process is called emissionemission
For absorption to occur, the energy of the For absorption to occur, the energy of the photon must exactly match an energy level photon must exactly match an energy level in the atom (or molecule) it contactsin the atom (or molecule) it contacts– EEphotonphoton = E = Eelectronic transitionelectronic transition
We distinguish two types of absorptionWe distinguish two types of absorption– AtomicAtomic– MolecularMolecular
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How Light Interacts with Matter.How Light Interacts with Matter.
Electrons bound to Electrons bound to atoms have atoms have discrete discrete energiesenergies (i.e. not all (i.e. not all energies are allowed).energies are allowed).
Thus, only photons of Thus, only photons of certain energy can certain energy can interact with the interact with the electrons in a given electrons in a given atom.atom.
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How Light Interacts with Matter.How Light Interacts with Matter.Consider hydrogen, the Consider hydrogen, the simplest atom.simplest atom.Hydrogen has a specific Hydrogen has a specific line spectrum.line spectrum.Each atom has its own Each atom has its own specific line spectrum specific line spectrum (atomic fingerprint).(atomic fingerprint).
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Energy Transitions and PhotonsEnergy Transitions and Photons
The energy of photon that can interact with a transition jump depends on the energy difference between the electronic levels.
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Unique Atomic SignaturesUnique Atomic SignaturesEach atom has a specific set of energy levels, and thus a unique set of photon wavelengths with which it can interact.
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Energy Level DiagramEnergy Level Diagram
Absorption and emission Absorption and emission for the sodium atom in the for the sodium atom in the gas phasegas phase
Illustrates discrete energy Illustrates discrete energy transfertransfer
ΔEtransition = E1 - E0 = hv hv = hc/hc/
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Molecular AbsorptionMolecular Absorption
More complex than atomic absorption More complex than atomic absorption because many more potential transitions because many more potential transitions existexist– Electronic energy levelsElectronic energy levels– Vibrational energy levelsVibrational energy levels– Rotational energy levelsRotational energy levels
EEmoleculemolecule = E = Eelectronicelectronic + E + Evibrationalvibrational + E + Erotationalrotational
– EEelectronicelectronic > E > Evibrationalvibrational > E > Erotationalrotational
Result - complex spectraResult - complex spectra
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Energy Level Diagram for Energy Level Diagram for Molecular AbsorptionMolecular Absorption
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Molecular Absorption Spectra of Molecular Absorption Spectra of Benzene in the Gas PhaseBenzene in the Gas Phase
Electronic Transition
Vibrational Transition Superimposed on the Electronic Transition
Absorption Band – A series of closely shaped peaks
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Molecular Molecular Absorption Absorption
Spectra in the Spectra in the Solution PhaseSolution Phase
In solvents the In solvents the rotational and rotational and vibrational transitions vibrational transitions are highly restricted are highly restricted resulting in resulting in broad broad bandband absorptionabsorption spectraspectra
Beer’s Lawor the Beer-Lambert Law
Pierre Bouguer discovered that light transmission decreases with the thickness of a transparent sample in 1729. Thislaw was later rediscovered by Lambert, a mathematician, and then by Beer, who published in 1852 what is now known asthe Beer-Lambert-Bouguer law. Beer's 1852 paper is the one that is often cited in older textbooks. Bouguer's contributionis rarely mentioned and the law is known as either "Beer's law" or "the Beer-Lambert law".
Spectroscopy Terms Describing Spectroscopy Terms Describing Absorption (Beer’s Law)Absorption (Beer’s Law)
Consider a beam of light Consider a beam of light with an (initial) radiant with an (initial) radiant intensity Pintensity Poo
The light passes through a The light passes through a solution of concentration (c)solution of concentration (c)The thickness of the The thickness of the solution is “b” cm. solution is “b” cm. The intensity of the light The intensity of the light after passage through the after passage through the solution (where absorption solution (where absorption occurs) is Poccurs) is P
P0hv P
b
Co
nce
ntr
atio
n (
c)
We DefineWe Define
Transmittance (T)Transmittance (T) = P/P = P/P0 0 (units = %)(units = %)
Absorbance (A)Absorbance (A) (units = none) (units = none)A = log (PA = log (P00/P)/P)
A = -log (T) = log (1/T)A = -log (T) = log (1/T)A = abc (or εbc) A = abc (or εbc) <---<--- Beer’s LawBeer’s Law
a = absorptivity (L/g cm)a = absorptivity (L/g cm)b = path length (cm)b = path length (cm)c = concentration (g/L)c = concentration (g/L)ε = molar absorptivity (L/mol cm) ε = molar absorptivity (L/mol cm)
– Used when concentration is in molar unitsUsed when concentration is in molar units
TransmittanceTransmittance
T => transmittanceT => transmittance
PPT = -----T = -----
PPoo
Po P
b
P0 = 10,000 P = 5,000
5.010000
5000
0
P
PT
-b-
ExampleExample
A = -log T = -log (0.5) = 0.3010
Beer’s LawBeer’s Law
A = abc =A = abc = bcbc
A
c
Beer’s LawBeer’s Law A = A = bbcc
Path Length Dependence, bPath Length Dependence, b
ReadoutAbsorbance
0.82
Source
Detector
Beer’s LawBeer’s Law A = A = bbcc
Path Length Dependence, bPath Length Dependence, b
ReadoutAbsorbance
0.62
Source
Detector
b
Sample
Beer’s LawBeer’s Law A = A = bbcc
Path Length Dependence, bPath Length Dependence, b
ReadoutAbsorbance
0.42
Source
Detector Samples
Beer’s LawBeer’s Law A = A = bbcc
Path Length Dependence, bPath Length Dependence, b
ReadoutAbsorbance
0.22
Source
Detector Samples
Beer’s LawBeer’s Law A = A = bcbc
Wavelength Dependence, aWavelength Dependence, a
ReadoutAbsorbance
0.80
Source
Detector
b
Beer’s LawBeer’s Law A = A = bcbc
Wavelength Dependence, aWavelength Dependence, a
ReadoutAbsorbance
0.82
Source
Detector
Beer’s LawBeer’s Law A = A = bcbc
Wavelength Dependence, aWavelength Dependence, a
ReadoutAbsorbance
0.30
Source
Detector
b
Beer’s LawBeer’s Law A = A = bcbc
Wavelength Dependence, aWavelength Dependence, a
ReadoutAbsorbance
0.80
Source
Detector
b
Non-Absorption LossesNon-Absorption Losses
"Reflection and "Reflection and scattering losses."scattering losses."
AKAAKA
The Guinness EffectThe Guinness Effect
Limitations to Beer’s LawLimitations to Beer’s Law
RealReal– At high concentrations charge distribution effects At high concentrations charge distribution effects
occur causing electrostatic interactions between occur causing electrostatic interactions between absorbing speciesabsorbing species
ChemicalChemical– Analyte dissociates/associates or reacts with solventAnalyte dissociates/associates or reacts with solvent
InstrumentalInstrumental– ε = f(λ); most light sources are polychromatic not ε = f(λ); most light sources are polychromatic not
monochromatic (small effect)monochromatic (small effect)– Stray light – comes from reflected radiation in the Stray light – comes from reflected radiation in the
monochromator reaching the exit slit.monochromator reaching the exit slit.
Chemical LimitationsChemical LimitationsA reaction is occurring as you record A reaction is occurring as you record
Absorbance measurementsAbsorbance measurements
CrCr22OO772-2- + H + H22O 2HO 2H++ + CrO + CrO44
2-2-
A550 A446
concentration concentrationwavelength
400 500300
CrO42-
Cr2O72-
Instrumental Limitations - ε = f(λ) Instrumental Limitations - ε = f(λ)
How/Why does ε How/Why does ε vary with λ?vary with λ?Consider a Consider a wavelength scan for wavelength scan for a molecular a molecular compound at two compound at two different wavelength different wavelength bandsbands
In reality, a In reality, a monochromator can monochromator can not isolate a single not isolate a single wavelength, but wavelength, but rather a small rather a small wavelength band wavelength band
Larger the Bandwidth – larger deviation
Instrumental Limitations – Stray LightInstrumental Limitations – Stray Light
How does stray light effect Absorbance How does stray light effect Absorbance and Beer’s Law?and Beer’s Law?
A = -log P/PA = -log P/Poo = log P = log Poo/P/P
When stray light (PWhen stray light (Pss) is present, the ) is present, the
absorbance observed (Aabsorbance observed (Aapparentapparent) is the sum ) is the sum
of the real (Aof the real (Arealreal) and stray absorbance ) and stray absorbance
(A(Astraystray))
Instrumental Limitations – Stray LightInstrumental Limitations – Stray Light
AAappapp = A = Arealreal + A + Astraystray = =
As the analyte concentration increases As the analyte concentration increases ([analyte]↑), the intensity of light exiting the ([analyte]↑), the intensity of light exiting the absorbance cell decreases (P↓)absorbance cell decreases (P↓)
Eventually, P < PEventually, P < Pss
s
so
P P
P P log
Instrumental Limitations – Stray LightInstrumental Limitations – Stray Light
Result – non-linear Result – non-linear absorption (Analyte absorption (Analyte vs. Conc.) as a vs. Conc.) as a function of analyte function of analyte concentrationconcentration– Similar to Similar to
polychromatic light polychromatic light limitationslimitations
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Emission of EMREmission of EMR
EMR is released when excited atoms or EMR is released when excited atoms or molecules return to ground statemolecules return to ground state– Reverse of the absorption processReverse of the absorption process– We call this process “We call this process “emissionemission””
Initial excitation can occur through a Initial excitation can occur through a number of pathwaysnumber of pathways– Absorption of EMRAbsorption of EMR– Electrical dischargeElectrical discharge– High temperatures (flame or arc)High temperatures (flame or arc)– Electron bombardmentElectron bombardment
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Emission of EMREmission of EMRWe distinguish several types of emissionWe distinguish several types of emission
1.1. AtomicAtomic2.2. X-RayX-Ray3.3. FluorescenceFluorescence
Involves moleculesInvolves moleculesResonance and non-resonance modesResonance and non-resonance modes
4.4. PhosphorescencePhosphorescenceNon-radiative relaxationNon-radiative relaxationSimilar to fluorescence only relaxation times are Similar to fluorescence only relaxation times are slower than fluorescenceslower than fluorescenceInvolves metastable intermediatesInvolves metastable intermediates
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Energy Level Diagrams and Energy Level Diagrams and EmissionEmission
Luminescence is the emission of light from any substance and occurs from electronically excited states.
It is formally divided into two categories: Molecular fluorescence. Molecular phosphorescence.
Its attractive feature is the inherent high sensitivity, 3 order of magnitude lower than absorption measurements (ppb).
Fluorescence is emission of light from excited singlet states (the electron in the excited state orbital is spin paired (has the opposite spin) to the electron in the ground state orbital) –therefore, return to the ground state is spin-allowed, and the excited state lifetime is short (1 -10 ns).
Phosphorescence is emission of light from excited triplet states (the electron in the excited orbital has the same spin orientation as the ground state electron) –therefore, the transition to the ground state is spin-forbidden, and the excited state lifetime is long (ms to seconds or even minutes!) Molecular chemiluminescence: emission from an excited species that formed in the course of chemical reaction.
Jablonski Diagram
Deactivation Processes
Intersystem Crossing: transition with spin change (e.g. S to T). As with internal conversion, the lowest singlet vibrational state overlaps one of upper triplet vibrational levels and a change in spin state is thus more probable.Intersystem crossing is most common in molecules that contain heavy atoms, such as iodine or bromine (the heavy-atom effect).
Fluorescence: emission not involving spin change (e.g. singlet→singlet),efficient, short-lived <10-5s.
Phosphorescence: emission involving spin change. Long-lived> 10-4s. A triplet →singlet transition is much less probable than singlet →singlet transition.
This transition may persist for some time after irradiation has been .discontinued since the average lifetime of the excited triplet state with respect to emission ranges from 10-4 to 10 s or more..
Dissociation: excitation to vibrational state with enough energy to break bond.Predissociation: relaxation to state with enough energy to break bond
Fluorescence Quenching
Quenching is ANY process that decreases the amount of fluorescence for a given number of input photons:
Collisional quenching –the excited state is de-activated via diffusional contact with a quencher (dynamic quenching)Fluorophores can form nonfluorescent complexes with quenchers. This process is referred to as static quenching since it occurs in the ground state and does not rely on diffusion or molecular collisions.attenuation of the emitted radiation by the fluorophore
methyl viologen
Collisional Quenching
How likely is fluorescence?
From the equation, it is clear that 0< φ< 1, and that a high value for kr and a small value for knr lead to the best quantum yield (i.e., fluorescence is faster than all other competing processes).
It should be noted that a change in quantum yield can occur owing to many factors (temperature, pH, solvent, presence of quenchers, dimerization, etc) and thus fluorescence intensity may not be directly proportional to concentration.
Molecular Luminescence Spectroscopy
S0-common, diamagnetic (not affected by B fields).
D0-unpaired electron, many radicals, two equal energy states.
T1-rare, paramagnetic (affected by B fields).
Energy (S1) > Energy (T1) (difference is energy required to flip electron spin).
Ground state Singlet, So
Ground state Doublet, Do
Excited state Triplet, T1
Excited state Singlet, S1
S1 So S1 So absorptionemission
What about Lifetimes?Absorption
S1S0 very fast 10 -15 -10 -13 s
RelaxationResonant emission S1 S0 fast 10 -9 -10 -5 s
(fluorescence) common in atoms
strong absorber - shorter lifetime
Non-resonant emission S1S0 fast 10 -9 -10 -5 s (fluorescence)
common in molecules, have extremely fast vibrational relaxation
red shifted emission (Stokes shift)
Stokes Shifting- The energy of the emission is typically less than that of absorption. Fluorescence typically occurs at lower energies or longer wavelengths. this is called Stokes Shifting.
Non-resonant emission T S0 slow 10 -5 -10 s (phosphorescence)
Transitions between states of different multiplicities are improbable (forbidden) (e.g. S or S)
Fluorescence Quantum Yield - ratio of number of molecules fluorescing to number excited.
What Affects the fluorescence quantum yield?(1) Excitation
Short l's break bonds increase kpre-dis and kdis
rarely observed most commonn
emission is usually from lowest lying excited state
(2) Lifetime of stateTransition probability measured by
Large implies short lifetime
Largest fluorescence from short lifetime/high state
n (10 -9 -10 -7 s > 10 -7 -10 -5 s)
(3) StructureFew conjugated aliphatics fluoresce
butMany aromatics fluoresce
Desire short lifetime S1, no/slowly accessible T1
Fluorescence increased by # fused rings and substitution on/in ring
Emission Intensity –the factors that control emission intensity include the presence of heteroatoms, presence of aromatic rings, overall structural rigidity, and resonance stabilization.
Presence of heteroatoms: often this can lead to unwanted π*→n transitions that are likely to convert to the triplet state, and give no fluorescence. All of the species shown below are non-fluorescent
(4) Rigidity
Rigid structures fluoresce
Increase in fluorescence with chelation