11

Instrumental Analysis Instrumental Analysis

Fundamentals of Fundamentals of SpectroscopySpectroscopy

22

(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)

33

Qualitative analysis: The spectrum

44

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

55

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?

66

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)

77

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.

88

The Electromagnetic SpectrumThe Electromagnetic Spectrum

99

1010

The Electromagnetic SpectrumThe Electromagnetic Spectrum

1111

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

1212

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)

1313

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

1414

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.

1515

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

1616

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

1717

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

1818

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

1919

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.

2020

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/

2121

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

2222

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.

2323

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).

2424

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.

2525

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.

2626

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/

2727

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

2828

Energy Level Diagram for Energy Level Diagram for Molecular AbsorptionMolecular Absorption

2929

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

3030

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

5252

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

5353

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

5454

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

Ethidium bromide