Here are some key points about the effects of multiple chromophores on absorption: - Additional chromophores in the same molecule cause a bathochromic (red) shift, moving the absorption maximum to longer wavelengths. This is due to increased conjugation and delocalization of the π electrons. - Absorption intensity (ε value) also increases with more chromophores, known as the hyperchromic effect. More π electrons means more efficient absorption. - Conjugated chromophores, where the π systems are connected, exhibit significant bathochromic and hyperchromic effects. The π* orbitals are delocalized over the whole conjugated system, lowering the energy gap for π-π*

1. MOLECULAR ABSORPTION
SPECTROSCOPY :
THEORY,
INSTRUMENTATION &
APPLICATION
CHAPTER 2

2. COMPONENTS OF
INSTRUMENTS FOR
OPTICAL SPECTROSCOPY

3. General Design of Optical
Instruments
Absorption
Emission

4. Five Basic Optical Instrument Components
1) Source – A stable source of radiant energy at the desired
wavelength (or  range).
2) Sample Container – A transparent container used to hold the
sample (cells, cuvettes, etc).
3) Wavelength Selector – A device that isolates a restricted
region of the EM spectrum used for measurement
(monochromators, prisms & filters).
4) Detector/Photoelectric Transducer – Converts the radiant
energy into a useable signal (usually electrical).
5) Signal Processor & Readout – Amplifies or attenuates the
transduced signal and sends it to a readout device as a
meter, digital readout, chart recorder, computer, etc.

5. I. Sources of Radiation
• Generate a beam of radiation that is stable and has
sufficient power.
A. Continuum Sources – emit radiation over a broad
wavelength range and the intensity of the radiation
changes slowly as a function of wavelength.
This type of source is commonly used in UV, visible
and IR instruments.
- Deuterium lamp is the most common UV source.
- Tungsten lamp is the most common visible source.
- Glowing inert solids are common sources for IR
instruments.

6. B. Line Sources – Emit a limited number lines or
bands of radiation at specific wavelengths.
- Used in atomic absorption spectroscopy
- Usually provide radiation in the UV and visible
region of the EM spectrum.
- Types of line source:
1) Hollow cathode lamps
2) Electrodeless discharge lamps
3) Lasers-Light – amplification by stimulated
emission of radiation

7. II. Wavelength Selectors
• Wavelength selectors output a limited,
narrow, continuous group of wavelengths
called a band.
• Two types of wavelength selectors:
1) Filters
2) Monochromators

8. A. Filters
- Two types of filters:
1) Interference Filters
2) Absorption Filters
B. Monochromators
- Wavelength selector that can continuously
scan a broad range of wavelengths
- Used in most scanning spectrometers
including UV, visible, and IR instruments.

9. III. Radiation Transducer (Detectors)
• Early detectors in spectroscopic
instruments were the human eye,
photographic plates or films. Modern
instruments contain devices that convert
the radiation to an electrical signal.
• Two general types of radiation transducers:
a. Photon detectors
b. Thermal detectors

10. A. Photon Detectors
- Commonly useful in ultraviolet, visible, and near
infrared instruments.
- Several types of photon detectors are available:
1. Vacuum phototubes
2. Photomultiplier tubes
3. Photovoltaic cells
4. Silicon photodiodes
5. Diode array transducers
6. Photoconductivity transducers

11. B. Thermal Detectors
- Used for infrared spectroscopy
because photons in the IR region lack
energy to cause photoemission of
electrons.
- Three types of thermal detectors:
1. Thermocouples
2. Bolometers
3. Pyroelectric transducers

12. IV. Sample Container
• Sample containers, usually called cells or cuvettes must have
windows that are transparent in the spectral region of interest.
• There are few types of cuvettes:
- quartz or fused silica
- silicate glass
- crystalline sodium chloride
Quartz or fused silica
- required for UV and may be used in visible region
Silicate glass
- Cheaper compared to quartz. Used in UV.
Crystalline sodium chloride
- Used in IR.

13.  Spectrometer
- is an instrument that provides information
about the intensity of radiation as a function
of wavelength or frequency.
 Spectrophotometer
- is a spectrometer equipped with one or
more exit slits and photoelectric transducers
that pemits the determination of the ratio of
the radiant power of two beams as a function
of wavelength as in absorption spectroscopy.

14. SUMMARY
Types of source, sample holder and detector
for various EM region
REGION SOURCE SAMPLE DETECTOR
HOLDER
Ultraviolet Deuterium lamp Quartz/fused silica Phototube, PM
tube, diode array
Visible Tungsten lamp Glass/quartz Phototube, PM
tube, diode array
Infrared Nernst glower Salt crystal e.g. Thermocouples,
(rare earth oxides crystal sodium bolometers
or silicon carbide chloride
glowers)

15. ULTRAVIOLET-VISIBLE
SPECTROSCOPY

16. In this lecture, you will learn:
• Absorption process in UV/VIS region in
terms of its electronic transitions
• Molecular species that absorb UV/VIS
radiation
• Important terminologies in UV/VIS
spectroscopy

17. INSTRUMENTATION
Important components in a UV-Vis spectrophotometer
5
1 2 3 4
Signal
Source Sample  selector Detector processor
lamp holder
& readout
UV region:
-Deuterium lamp; Quartz/fused silica Prism/monochromator Phototube,
H2 discharge tube PM tube, diode
array
Visible region:
- Tungsten lamp Glass/quartz Prism/monochromator Phototube,
PM tube, diode
array

18. Instrumentation
• UV-Visible instrument
1. Single beam
2. Double beam

19. Single beam instrument

20. • Single beam instrument
- One radiation source
- Filter/monochromator ( selector)
- Cells
- Detector
- Readout device

21. Single beam instrument
• Disadvantages:
– Two separate readings has to be made on the
light. This result in some error because the
fluctuations in the intensity of the light do
occur in the line voltage, the power source
and in the light bulb btw measurements.
– Changing of wavelength is accompanied by a
change in light intensity. Thus spectral
scanning is not possible.

22. Double beam instrument
Double-beam instrument with beams separated in space

23. • Double-beam instrument
Advantages:
1. Compensate for all but most short-term
fluctuations in the radiant output of the
source as well as for drift in the
transducer and amplifier.
2. Compensate for wide variations in
source intensity with .
3. Continuous recording of transmittance
or absorbance spectra.

24. ORGANIC INORGANIC
COMPOUNDS SPECIES
MOLECULAR SPECIES THAT
ABSORB UV/VISIBLE RADIATION
CHARGE
TRANSFER

25. Definitions:
• Organic compounds
– Chemical compound whose molecule contain carbon
– E.g. C6H6, C3H4
• Inorganic species
– Chemical compound that does not contain carbon.
– E.g. transition metal, lanthanide and actinide elements.
– Cr, Co, Ni, etc
• Charge transfer
– A complex where one species is an electron donor and
the other is an electron acceptor.
– E.g. iron (III) thiocyanate complex

26. PERIODIC TABLE OF
ELEMENTS

27. ULTRAVIOLET-VISIBLE
SPECTROSCOPY
• In UV/VIS spectroscopy, the transitions
which result in the absorption of EM
radiation in this region are transitions
between electronic energy levels.

28. Molecular absorption
• In molecules, not only have electronic
level but also consists of vibrational and
rotational sub-levels.
• This result in band spectra.

29. Types of transitions
• 3 types of electronic transitions
- ,  and n electrons
- d and f electrons
- charge transfer electrons

30. What is σ,  and n electrons?
single covalent bonds (σ)
H + O + H H O H or H O H
lone pairs(n)
O C O or O C O
double bonds ()
N N or N N
triple bond ()

31. Sigma () electron
 Electrons involved in single bonds such as
those between carbon and hydrogen in alkanes.
 These bonds are called sigma () bonds.
 The amount of energy required to excite
electrons in  bond is more than UV photons of
wavelength. For this reason, alkanes and other
saturated compounds (compounds with only
single bonds) do not absorb UV radiation and
therefore frequently very useful as transparent
solvents for the study of other molecules. For
example, hexane, C6H14.

32. Pi () electron
• Electrons involved in double and triple
bonds (unsaturated).
• These bonds involve a pi () bond.
• For exampel: alkenes, alkynes,conjugated
olefins and aromatic compounds.
• Electrons in  bonds are excited relatively
easily; these compounds commonly
absorb in the UV or visible region.

33. • Examples of organic molecules containing
 bonds.
H
CH2CH3 CH3 C C H
H C H
propyne
C C
C C
H C H H H
H H C C
ethylbenzene
benzene C C H
H H
1,3-butadiene

34. n electron
• Electrons that are not involved in
bonding between atoms are called n
electrons.
• Organic compounds containing nitrogen,
oxygen, sulfur or halogens frequently
contain electrons that re nonbonding.
• Compounds that contain n electrons
absorb UV/VIS radiation.

35. • Examples of organic molecules with non-
bonding electrons.
..
: NH2 O:
C
R H3C H
C C
..
: Br
.. H
aminobenzene Carbonyl compound
If R = H  aldehyde 2-bromopropene
If R = CnHn  ketone

36. ABSORPTION BY ORGANIC COMPOUNDS
• UV/Vis absorption by organic compounds
requires that the energy absorbed
corresponds to a jump from occupied
orbital to an unoccupied orbital of greater
energy.
• Generally, the most probable transition is
from the highest occupied molecular
orbital (HOMO) to the lowest unoccupied
molecular orbital (LUMO).

37. Electronic energy levels diagram
* Antibonding
Unoccupied levels
* Antibonding
  *
n  *
n  *
  *
Energy
n Nonbonding
 Bonding
Occupied levels
 Bonding

38. Electronic transitions
  * In alkanes
  * In alkenes, carbonyl compounds, alkynes, azo
compounds
Increasing
energy n  * In oxygen, nitrogen, sulfur and halogen
compounds
n  * In carbonyl compounds

39. Electronic transitions
 * transitions
• The energy required to induce a  *
transition is large (see the arrow in energy
level diagram).
• Never observed in the ordinarily accessible
ultraviolet region.
• This type of absorption corresponds to
breaking of C-C, C-O, C-H, C-X, ….bonds

40. n * transitions
- Saturated compounds containing atoms with unshared
electron pairs (non-bonding electrons).
- Compounds containing O, S, N and halogens can
absorb via this type of transition.
- Absorption are typically in the  range, 150 - 250 nm
region and are not very intense.
-  range: 100 – 3000 cm-1mol-1
- Absorption maxima tend to shift to shorter  in polar
solvents.
e.g. H2O, CH3CH2OH

41. Some examples of absorption due to
n * transitions
Compound max (nm) max
H2O 167 1480
CH3OH 184 150
CH3Cl 173 200
CH3I 258 365
(CH3)2O 184 2520
CH3NH2 215 600

42. n * transitions
- Unsaturated compounds containing atoms
with unshared electron pairs (nonbonding
electrons)
- These result in some of the most intense
absorption in  range, 200 – 700 nm
- Unsaturated functional group
- to provide the  orbitals
-  range: 10 – 100 Lcm-1mol-1

43.  * transitions
- Compounds with unsaturated functional
groups to provide the  orbitals.
- These result in some of the most intense
absorption in  range, 200 – 700 nm
-  range: 1000 – 10,000 Lcm-1mol-1

44. Examples n * and  *
H O
H C C
H H
  * at 180 nm
n  * at 290 nm

45. MOLECULAR SPECIES THAT ABSORB
UV/VISIBLE RADIATION
(A) Absorption by organic compounds
2 types of electrons are responsible:
i. Shared electrons that participate directly
in bond formation ( and  bonding
electrons)
ii. Unshared outer electrons (nonbonding
or n electrons)

46. Absorption by organic compounds
• The shared electrons in single bonds, C-C or
C-H ( electrons) are so firmly held.
Therefore, not easily excited to higher E
levels. Absorption (  *) occurs only in the
vacuum UV region (  180 nm).
• Electrons in double & triple bonds (electrons)
are more loosely held. Therefore, more easily
excited by radiation. Absorptions (   *) for
species with unsaturated bonds occur in the
UV/VIS region (   180 nm)

47. Absorption by organic compounds
CHROMOPHORES
Unsaturated organic functional
groups that absorb in the UV/VIS
region.

48. Typical organic functional groups
that serve as chromophores
Chromophores Chemical structure Type of transition
Acetylenic -CC-   *
Amide -CONH2   *, n  *
Carbonyl >C=O   *, n  *
Carboxylic acid -COOH   *, n  *
Ester -COOR   *, n  *
Nitro -NO2   *, n  *
Olefin >C=C<   *

50. Absorption by organic compounds
AUXOCHROME
• Groups such as –OH, -NH2 & halogens
that attached to the double bonded atoms
cause the normal chromophoric absorption
to occur at longer  (red shift).

51. Effect of Multichromophores
on Absorption
• More chromophores in the same molecule
cause bathochromic effect ( shift to longer )
and hyperchromic effect (increase in
intensity).
• In conjugated chromophores, * electrons are
delocalized over larger number of atoms.
This cause a decrease in the energy of  *
transitions and an increase in  due to an
increase in probability for transition.

52. Absorption by organic compounds
• Factors that influenced the :
i) Solvent effects (shift to shorter : blue
shift)
ii) Structural details of the molecules

53. Absorption spectra for typical organic
compounds

54. Important terminologies
• Hypsochromic shift (blue shift)
- Absorption maximum shifted to shorter 
• Bathochromic shift (red shift)
- Absorption maximum shifted to longer 

55. Terminology for Absorption Shifts
Nature of Shift Descriptive Term
To Longer Wavelength Bathochromic
To Shorter Wavelength Hypsochromic
To Greater Absorbance Hyperchromic
To Lower Absorbance Hypochromic

56. (B) Absorption by inorganic species
• Involving d and f electrons absorption
• 3d & 4d electrons
- 1st and 2nd transition metal series
e.g. Cr, Co, Ni & Cu
- Absorb broad bands of VIS radiation
- Absorption involved transitions between
filled and unfilled d-orbitals with energies that
depend on the ligands, such as Cl-, H2O, NH3
or CN- which are bonded to the metal ions.

57. Absorption spectra of some transition-metal ions and rare
earth ions
Most transition metal ions are colored (absorb in UV-VIS) due to d  d
electronic transitions

58. Absorption by inorganic species
• 4f & 5f electrons
- Ions of lanthanide and actinide elements
- Their spectra consists of narrow, well-
defined characteristic absorption peaks.

59. (C) Charge transfer absorption
 Absorption involved transfer of electron from
the donor to an orbital that is largely
associated with the acceptor.
 an electron occupying in a  or  orbital
(electron donor) in the ligand is transferred to
an unfilled orbital of the metal (electron
acceptor) and vice-versa.
 e.g. red colour of the iron (III) thiocyanate
complex

60. Absorption spectra of aqueous charge transfer
complexes

61. Quantitative Analysis
• The fundamental law on which absorption
methods are based on Beer’s Law (Beer-
Lambert Law).

62. Measuring Absorbance
• You must always attempt to work at the
wavelength of maximum absorbance
(max).
• This is the point of maximum response, so
better sensitivity and lower detection limits.
• You will also have reduced error in your
measurement.

64. Quantitative Analysis
• Calibration curve method
• Standard addition method

65. • Calibration curve method
- A general method for determining the
concentration of a substance in an
unknown sample by comparing the
unknown to a set of standard sample of
known concentration.

66. Standard Calibration Curve
Absorbance
How to measure the concentration of unknown?
• Practically, you have measure the absorbance of your
unknown. Once you know the absorbance value, you can just
read the corresponding concentration from the graph.

67. How to produce standard calibration curve
Absorbance
• Prepare a series of
standard solution with
known concentration.
• Measure the absorbance of Calibration standard
the standard solutions.
• Plot the graph Abs vs
concentration of std.
• Find the “best’ straight line.
Stock solution
100 ppm

68. • The slope of the line, m:
m = y2 – y1
x2 – x1
• The intercept, b:
b = y – mx
• Thus, the equation for the least-square line is:
y = mx + b

69. Concentration, x y = mx + b
5
10
15
20
25
• From the least-square line equation, you can calculate
the new y values by substituting the x value.
• Then plot the graph.

70. Standard addition method
- used to overcome matrix effect
- involves adding one or more increments
of a standard solution to sample aliquots
of the same size.
- Each solution is diluted to a fixed volume
before measuring its absorbance.

71. Standard Addition Plot
Absorbance

72. How to produce standard
addition curve?
1. Add same quantity of unknown sample to a series of flasks.
2. Add varying amounts of standard (made in solvent) to each
flasks, e.g. 0, 5, 10, 15 mL).
3. Fill each flask to line, mix and measure.

73. Standard Addition
Methods
Single-point standard Multiple standard
addition method addition method

74. Standard addition
- if Beer’s Law is obeyed,
A = bVstdCstd + bVxCx
Vt Vt
= kVstdCstd + kVxCx
k is a constant equal to  b
Vt

75. Standard Addition
- Plot a graph: A vs Vstd
A = mVstd + b
where the slope m and intercept b are:
m = kCstd ; b = kVxCx

76. • Cx can be obtained from the ratio of these
two quantities: m and b
b = kVxCx
m kCstd
Cx = bCstd
mVx

77. Example:
• 10 ml aliquots of raw-water sample were
pipetted into 50.0 ml volumetric flasks. Then,
0.00, 5.00, 10.00, 15.00 and 20.00 ml
respectively of a standard solution containing
10 ppm of Fe3+ were added to the flasks,
followed by an excess of aqueous potassium
thiocyanate in order to produce the red iron-
thiocyanate complex. All the resultant
solutions were diluted to volume and the
absorbance of each solution was measured
at the same.

78. The results obtained:
Vol. of std added Absorbance
(ml) (A)
0 0.215
5.00 0.424
10.00 0.625
15.00 0.836
20.00 1.040
Calculate the concentration of Fe3+ (in ppm)
in the raw-water sample

79. Absorbance vs Vol. of std added
1.2
1
0.8
Absorbance
b = 0.24
0.6
Slope, m = 0.0382
(Vstd)0 = -6.31 ml 0.4
0.2
0
-10 -5 0 5 10 15 20 25
Vol. of std
Note: From the graph, extrapolated value represents the volume of
reagent corresponding to zero instrument response.

80. • The unknown concentration of the analyte
in the solution is then calculated:
Csample = -(Vstd)0Cstd
Vsample
Cx = bCstd
mVx

81. SELF-EXERCISE
The chromium in an aqueous sample was determined by pipetting
10.0 ml of the unknown into each of 50.0 mL volumetric flasks.
Various volumes of a standard containing 12.2 ppm Cr were added
to the flasks, following which the solutions were diluted to the mark.
Volume of Volume of Absorbance
unknown (mL) standard (mL)
10.0 0.0 0.201
10.0 10.0 0.292
10.0 20.0 0.378
10.0 30.0 0.467
10.0 40.0 0.554
i) Plot a suitable graph to determine the concentration of Cr in the
aqueous sample.

82. Visible Spectroscopy
The portion of the EM spectrum from 400-800 is
observable to humans- we (and some other mammals)
have the adaptation of seeing color at the expense of
greater detail.
400 500 600 700 800
, nm
Violet 400-420
Indigo 420-440
Blue 440-490
Green 490-570
Yellow 570-585
Orange 585-620
Red 620-780

83. Visible Spectroscopy
When white (continuum of λ)
light passes through, or is
reflected by a surface, those λs
that are absorbed are
removed from the transmitted
or reflected light respectively.
What is “seen” is the
complimentary colors (those
that are not absorbed).
This is the origin of the “color
wheel”.

84. Visible Spectroscopy
Organic compounds that are “colored” are typically those with
extensively conjugated systems (typically more than five).
Consider b-carotene.
b-carotene, max = 455 nm
λmax is at 455 nm – in the far blue
region of the spectrum . This is
absorbed.
The remaining light has the
complementary color of orange.

85. Visible Spectroscopy
lycopene, max = 474 nm
O
H
N
N
H
O
indigo
λmax for lycopene is at 474 nm – in the near blue region of
the spectrum this is absorbed, the compliment is now red.
λmax for indigo is at 602 nm – in the orange region of the
spectrum. This is absorbed, the compliment is now indigo!