1. Instrumental Analysis
Fundamentals of Spectroscopy

2. (Absorption) Spectrophotometry n General Stuff:
•Qualitative: Spectrum (a plot of A vs. l) is
characteristic of a specific species
•Quantitative: Absorbance at a particular l can be
related to the amount of absorbing species
Definitions and units
l .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)

3. Qualitative analysis: The spectrum

4. Molecular and Atomic Spectrometry
Spectrometry is the study of electromagnetic radiation (EMR) and its applications
To begin to understand the theory and instrumental application of spectrometry requires an understanding of the interaction of EMR (i.e. light) with matter

5. Questions What is nature of light? Are their different types of light?
How are they the same?
How are they different?
How does light propagate?

6. What is Light? Light is a form of energy
Light travels through space at extremely high velocities
The speed of light (c) ~ 3 x 1010 cm/sec or 186,000 miles per second
Light is classified as electromagnetic radiation (EMR)

7. Characteristics of Light
Light behaves like a wave.
That is, it can be modeled or characterized with wave like properties.
Light also behaves like a particle.
The photon and photoelectric effect.
Today, we envision light as a self-contained packet of energy, a photon, which has both wave and particle like properties.

8. The Electromagnetic Spectrum

10. The Electromagnetic Spectrum

11. The EMR Spectrum
Different portions of the EMR spectrum and different types of spectroscopy involve different parts (quantum states) of the atom

12. EMR Wave Characteristics
Wavelength (l) is the distance from one wave crest to the next.
Amplitude is the vertical distance from the midline of a wave to the peak or trough.
Frequency (v) is the number of waves that pass through a particular point in 1 second (Hz = 1 cycle/s)

13. EMR Wave Characteristics
The frequency of a wave is dictated (or fixed) by its source, it doesn’t change as the wave passes through different mediums.
The speed of a wave (u), however, can change as the medium through which it travels changes
umedium = v = c/n
Where n = refractive index
nvacuum = 1
nair = (vair = c)
nglass ~1.5 (vgas ~ 0.67c)
Since v is fixed, as  decreases, u must also decrease

14. Wave Properties of Electromagnetic Radiation
EMR has both electric (E) and magnetic (H) components that propagate at right angles to each other.

15. Particle Properties of EMR
The energy of a photon depends on its frequency (v)
Ephoton = hv
h = Planck’s constant
h = 6.63 x erg sec or 6.63 x Js

16. Relationship between Wave and Particle Properties of EMR
Ephoton = hv ; umedium = v = c/n
With these two relationships, if you know one of the following, you can calculate the other two
Energy of photon
Wavelength of light
Frequency of light
Ephoton =
Ephoton = hv ; umedium = v = c/n
With these two relationships, if you know one of the following, you can calculate the other two
Energy of photon
Wavelength of light
Frequency of light
Ephoton =

17. Relationship between Wave and Particle Properties of EMR
Example: What is the energy of a 500 nm photon?
= c/l = (3 x 108 m s-1)/(5.0 x 10-7 m)
n = 6 x 1014 s-1
E = hn =(6.626 x J•s)(6 x 1014 s-1) = 4 x J

18. How Light Interacts with Matter.
Atoms are the basic blocks of matter.
They consist of heavy particles (called protons and neutrons) in the nucleus, surrounded by lighter particles called electrons

19. How Light Interacts with Matter.
An electron will interact with a photon.
An electron that absorbs a photon will gain energy.
An electron that loses energy must emit a photon.
The total energy (electron plus photon) remains constant during this process.

20. Characteristics of Absorption
Absorption is defined as the process by which EMR is transferred, in the form of energy, to the medium (s, l, or g) through which it is traveling
Involves discrete energy transfers
Frequency and wavelength selective
Ephoton = hv = c/

21. Characteristics of Absorption
Involves transitions from ground state energy levels to “excited” states
The reverse process is called emission
For absorption to occur, the energy of the photon must exactly match an energy level in the atom (or molecule) it contacts
Ephoton = Eelectronic transition
We distinguish two types of absorption
Atomic
Molecular

22. How Light Interacts with Matter.
Electrons bound to atoms have discrete energies (i.e. not all energies are allowed).
Thus, only photons of certain energy can interact with the electrons in a given atom.

23. How Light Interacts with Matter.
Consider hydrogen, the simplest atom.
Hydrogen has a specific line spectrum.
Each atom has its own specific line spectrum (atomic fingerprint).

24. Energy Transitions and Photons
The energy of photon that can interact with a transition jump depends on the energy difference between the electronic levels.

25. Unique Atomic Signatures
Each atom has a specific set of energy levels, and thus a unique set of photon wavelengths with which it can interact.

26. Energy Level Diagram
Absorption and emission for the sodium atom in the gas phase
Illustrates discrete energy transfer
ΔEtransition = E1 - E0 = hv = hc/

27. Molecular Absorption
More complex than atomic absorption because many more potential transitions exist
Electronic energy levels
Vibrational energy levels
Rotational energy levels
Emolecule = Eelectronic + Evibrational + Erotational
Eelectronic > Evibrational > Erotational
Result - complex spectra

28. Energy Level Diagram for Molecular Absorption

29. Molecular Absorption Spectra of Benzene in the Gas Phase
Electronic Transition
Vibrational Transition Superimposed on the Electronic Transition
Absorption Band – A series of closely shaped peaks

30. Molecular Absorption Spectra in the Solution Phase
In solvents the rotational and vibrational transitions are highly restricted resulting in broad band absorption spectra

31. or the Beer-Lambert Law
Beer’s Law
or the Beer-Lambert Law
Pierre Bouguer discovered that light transmission decreases with the thickness of a transparent sample in This
law was later rediscovered by Lambert, a mathematician, and then by Beer, who published in 1852 what is now known as
the Beer-Lambert-Bouguer law. Beer's 1852 paper is the one that is often cited in older textbooks. Bouguer's contribution
is rarely mentioned and the law is known as either "Beer's law" or "the Beer-Lambert law".

32. Spectroscopy Terms Describing Absorption (Beer’s Law)
Consider a beam of light with an (initial) radiant intensity Po
The light passes through a solution of concentration (c)
The thickness of the solution is “b” cm.
The intensity of the light after passage through the solution (where absorption occurs) is P
Concentration (c) P0 hv P b

33. We Define Transmittance (T) = P/P0 (units = %)
Absorbance (A) (units = none)
A = log (P0/P)
A = -log (T) = log (1/T)
A = abc (or εbc) <--- Beer’s Law
a = absorptivity (L/g cm)
b = path length (cm)
c = concentration (g/L)
ε = molar absorptivity (L/mol cm)
Used when concentration is in molar units

34. Transmittance
T => transmittance P
T = ----- Po b Po P

35. Example
P0 = 10,000
P = 5,000
-b-
A = -log T = -log (0.5) =

36. Beer’s Law
A = abc = ebc A c

37. Beer’s Law A = ebc Path Length Dependence, b Readout Absorbance 0.82
Source
Detector

38. Beer’s Law A = ebc Path Length Dependence, b Readout Absorbance 0.62
Source b
Detector
Sample

39. Beer’s Law A = ebc Path Length Dependence, b Readout Absorbance 0.42
Source
Detector
Samples

40. Beer’s Law A = ebc Path Length Dependence, b Readout Absorbance 0.22
Source
Detector
Samples

41. Beer’s Law A = ebc Wavelength Dependence, a Readout Absorbance 0.80
Source b
Detector

42. Beer’s Law A = ebc Wavelength Dependence, a Readout Absorbance 0.82
Source
Detector

43. Beer’s Law A = ebc Wavelength Dependence, a Readout Absorbance 0.30
Source b
Detector

44. Beer’s Law A = ebc Wavelength Dependence, a Readout Absorbance 0.80
Source b
Detector

45. Non-Absorption Losses
"Reflection and scattering losses."
AKA
The Guinness Effect

46. Limitations to Beer’s Law
Real
At high concentrations charge distribution effects occur causing electrostatic interactions between absorbing species
Chemical
Analyte dissociates/associates or reacts with solvent
Instrumental
ε = f(λ); most light sources are polychromatic not monochromatic (small effect)
Stray light – comes from reflected radiation in the monochromator reaching the exit slit.

47. Chemical Limitations
A reaction is occurring as you record Absorbance measurements
Cr2O H2O H CrO42-
CrO42-
Cr2O72-
A550
A446
300
400
500
concentration
concentration
wavelength

48. Instrumental Limitations - ε = f(λ)
How/Why does ε vary with λ?
Consider a wavelength scan for a molecular compound at two different wavelength bands
In reality, a monochromator can not isolate a single wavelength, but rather a small wavelength band
Larger the Bandwidth – larger deviation

49. Instrumental Limitations – Stray Light
How does stray light effect Absorbance and Beer’s Law?
A = -log P/Po = log Po/P
When stray light (Ps) is present, the absorbance observed (Aapparent) is the sum of the real (Areal) and stray absorbance (Astray)

50. Instrumental Limitations – Stray Light
Aapp = Areal + Astray =
As the analyte concentration increases ([analyte]↑), the intensity of light exiting the absorbance cell decreases (P↓)
Eventually, P < Ps

51. Instrumental Limitations – Stray Light
Result – non-linear absorption (Analyte vs. Conc.) as a function of analyte concentration
Similar to polychromatic light limitations

52. Emission of EMR
EMR is released when excited atoms or molecules return to ground state
Reverse of the absorption process
We call this process “emission”
Initial excitation can occur through a number of pathways
Absorption of EMR
Electrical discharge
High temperatures (flame or arc)
Electron bombardment

53. Emission of EMR We distinguish several types of emission Atomic X-Ray
Fluorescence
Involves molecules
Resonance and non-resonance modes
Phosphorescence
Non-radiative relaxation
Similar to fluorescence only relaxation times are slower than fluorescence
Involves metastable intermediates

54. Energy Level Diagrams and Emission

55. 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).

56. 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.

57. Jablonski Diagram

58. 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

59. 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

60. Collisional Quenching
methyl viologen

62. 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.

63. 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
emission
absorption
S So S So

64. Absorption Relaxation
What about Lifetimes?
Absorption
S1S0 very fast s
Relaxation
Resonant emission S1 S0 fast s (fluorescence)
common in atoms
strong absorber - shorter lifetime
Non-resonant emission S1S0 fast s (fluorescence)
common in molecules, have extremely fast vibrational relaxation
red shifted emission (Stokes shift)

65. 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.

66. Non-resonant emission T1 S0 slow 10 -5 -10 s (phosphorescence)
Transitions between states of different multiplicities are improbable (forbidden)
(e.g. T S or T S)

67. Fluorescence Quantum Yield - ratio of number of molecules fluorescing to number excited.

68. What Affects the fluorescence quantum yield? (1) Excitation l
Short l's break bonds increase kpre-dis and kdis
rarely observed most common
s* ®s p* ® n p* ® p
emission is usually from lowest lying excited state
(2) Lifetime of state
Transition probability measured by e
Large e implies short lifetime
Largest fluorescence from short lifetime/high e state
p*®p > p*® n ( s > s)

69. Few conjugated aliphatics fluoresce but Many aromatics fluoresce
(3) Structure
Few conjugated aliphatics fluoresce
but
Many 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

70. (4) Rigidity
Rigid structures fluoresce
Increase in fluorescence with chelation

71. Ethidium bromide