This document discusses spectroscopy techniques, specifically atomic absorption spectroscopy (AAS). It begins by explaining the electromagnetic spectrum and photon interactions with matter. It then describes different types of spectroscopy including absorption, fluorescence, and phosphorescence. Beer-Lambert law is introduced which states that absorbance is directly proportional to concentration. Instrumental components of a spectrophotometer such as light sources, monochromators, sample holders, and detectors are outlined. The principles of atomic absorption spectroscopy are explained including hollow cathode lamps and atomic excitation. Advantages of AAS compared to atomic emission spectroscopy are provided.

1. Spectroscopy and AAS
Dr. Aparna khandelwal

4. The electromagnetic spectrum

5. The photon is the elementary particle
responsible for electromagnetic phenomena
• As a particle, it interacts with matter by transferring its energy E:
• E= hν = hc/λ
• h - Planck constant (h = 6.63 x 1034 Js)
• ν - frequency of the radiation (no. of wave cycle pass through a
point in one sec.
• c – speed of light

6. Types of spectroscopy
• When radiation meets matter,the radiation is either
Scattered
emitted
or absorbed
• This gives rise to three principle branches of spectroscopy
• After absorption of light molecule from excited statecan return to ground
stateby means of radioactive transition, energy is reemitted in the form
of luminescence.

7. Absorption spectroscopy
• Absorption occurs only when energy of radiation matches the
difference between two energy levels.
• Also, type of transition depends on the energy of electromagnetic
radiation.

8. Effect of EM radiation on interaction of matter
depends on energy associated with the radiation

9. Molecular orbitals
• Different orbitals combine to yield molecular orbitals that generally fall into
one of the five different classes
• s orbitals combine to form------ binding s and the antibinding s* orbitals.
• Some p orbitals combine to form----- binding p and the anti-binding
p*orbitals.
• Other p orbitals combine to form ------non-binding n orbitals.
• The population of binding orbitals strengthens a chemical bond, and, vice
versa, the population of anti-binding orbitals weakens a chemical bond

10. Energy scheme for molecular orbitals
Arrows indicate possible electronic transitions.
The length of the arrows indicates the energy required to be put into the system in
order to enable the transition.

11. Spin and multiplicity
• For paired electrons in one orbital,
normally have antiparallel spin
• S= +½ + -½ = 0
• The multiplicity is thus
M=2 x 0 + 1 = 1.
Such a state is thus called a singlet state
and denotated as ‘S’.
The ground state of a molecule is a
singlet state, S0
Excited singlet statesare S1, S2 etc
• The total spin S is calculated from the
individual electron spins.
• S = spin(electron 1)+ spin(electron 2)
• The multiplicity M is obtained by​
M= 2xS+1
• In case the spins of both electrons
are oriented in a parallel fashion
• S=+½ + +½=1
• The multiplicity is thus
M=2 x 1 + 1 = 3.
Such a stateis thus called a
Triplet stateand denotated as ‘T’.
This stateis one of the excited state
only

12. Jablonski diagram

13. For UV-Visible region
In order to absorb EM radiationin this range,
Moleculemust contain
Such molecules known
as chromophores

14. In protein there are 3 types of chromophores
• Peptide bonds
• certain amino acid side chains( tyrosine and tryptphan mainly)
• Certain prosthetic groups and coenzymes( e.g.porphyrine groups such
as in haem)

15. Beer Lambert law
• Lambert's law states that when monochromatic light passes through a
transparent medium,

16. Beer Lambert law
• Beer's law states that intensity of transmitted monochromatic light
decreases exponentially as the concentration of absorbing substance
increases

17. Mathematical expression of lambert Beer's
law

18. Deviation
According to the Beer–Lambert law
1. This might not be the case any more in samples with high
absorbance. chromophores might dimerise at high concentrations
2. Every spectrophotometer has a certain amount of stray light, which
is light received at the detector but not anticipated in the spectral band
isolated by the monochromator.
• In order to obtain reasonable signal-to-noise ratios, the intensity of
light at the chosen wavelength should be 10 times higher than the
intensity of the stray light.

19. spectrophotometer
• An instrument used to measure the absorbance by measuring the
amount of light of a given wavelength that is transmitted by a sample
is termed spectrophotometer.

20. • Light source
• Hydrogen and deuterium lamp
• Xenon discharge lamps
• Mercury arc
• Tungsten filament
• Lasers

21. Laser
• LIGHT AMPLIFICATION BY STIMULATED EMMISION OF RADIATION

22. obviates any problems of variation in light intensity, as both referenceand
sample would be affected equally
one detector to measures the incoming and the
transmitted intensity alternately is better, so that it
eliminate the differencein the potential variations .
series of engraved fine lines.
The distance between the lines decides
the magnitude of wavelength diffracted
By varying the distance between the
lines, different wavelengths are selected

23. DIFFERACTION GRATING

24. borosilicate glass and normal
plastics absorbUV light
such cuvettes can only be used
for applications in the visible
range of the spectrum (up to
350 nm).
For UV measurements, quartz
cuvettes are used.
However, disposable plastic
cuvettes have been developed
that allow for measurements
over the entire range of the
UV/Vis spectrum

25. Photodetectors
• Converts light into electric signal
• Most commonly used PD in UV-Visible range is PMT
• PMT contains
• A cathode
• A light sensetive metal
• A series of dynodes
No. Of photons striking to
its photosensitivesurface

26. Characteristic of PMT
• Have rapid response times
• Very sensetive
• Slow to fatigue
• Should be carefully shielded from all stray light
• It may burn out if exposed to room light with voltage applied
• When voltage is applied to a PMT in the absence of any incident light
some current is usually produced k/a DARK CURRENT
• It is desirable to have dark current of a PMT at its lowest level
because this current appear as background noise

27. Signal to noise ratio Measure of the power of desired signal relativeto the
background signal
• Expressed in decibles.
• Should be higher than 1:1 or 0 db
• Greater the SNR better the resolution

28. Atomic absorption spectrophotometry
• We discussed the general theory of electronic transitions and said
that molecules give rise to band spectra, but atoms yield clearly
defined line spectra.
• In atomic emission spectroscopy these lines can be observed as light
of a particular wavelength (colour).
• Conversely, black lines can be observed against a bright background in
atomic absorption

29. Principle
• In a spectrum of an element, the absorption or emission
wavelengths are associated with transitions that require a minimum
of energy change.
(‘D-line’) due to the transitionof an electron
from the 3s to the 3p orbital and return

30. AAS vs AES

31. AAS
Hollow cathode lamp
Cathode made up of
metal of intrest
Filled with inert gas
On applying voltage its atom
ionises & strike to cathode
and excite the atoms of
cathode
Sample droplet
vaporises and absorb
energy and gets excited

32. Atomic emission spectroscopy

33. AES

34. • The energy absorbed or emitted is proportional to the number of
atoms in the optical path.
• Concentration determination with AES or AAS is carried out by
comparison with calibration standards

35. AAS vs AES
AAS
• More accurate
• More sensitive
• Quantitative assesment
• Intensity of light absorbed
AES
• Faster
• Qualitative assesment
• Intensity of light emitted is
studied