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MOLECULAR ABSORPTION
SPECTROSCOPY :
THEORY,
INSTRUMENTATION &
APPLICATION
CHAPTER 2
COMPONENTS OF
INSTRUMENTS FOR
OPTICAL SPECTROSCOPY
General Design of Optical
Instruments
Absorption
Emission
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.
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.
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
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
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.
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
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
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
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.
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.
SUMMARY
Types of source, sample holder and detector
for various EM region
REGION SOURCE SAMPLE
HOLDER
DETECTOR
Ultraviolet Deuterium lamp Quartz/fused silica Phototube, PM
tube, diode array
Visible Tungsten lamp Glass/quartz Phototube, PM
tube, diode array
Infrared Nernst glower
(rare earth oxides
or silicon carbide
glowers)
Salt crystal e.g.
crystal sodium
chloride
Thermocouples,
bolometers
ULTRAVIOLET-VISIBLE
SPECTROSCOPY
• 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
In this lecture, you will learn:
INSTRUMENTATION
Important components in a UV-Vis spectrophotometer
Source
lamp
Sample
holder selector Detector
Signal
processor
& readout
1 2 3 4 5
UV region:
-Deuterium lamp;
H2 discharge tube
Visible region:
- Tungsten lamp
Glass/quartz
Prism/monochromator
Phototube,
PM tube, diode
array
Quartz/fused silica
Prism/monochromator
Phototube,
PM tube, diode
array
Instrumentation
• UV-Visible instrument
1. Single beam
2. Double beam
Single beam instrument
• Single beam instrument
- One radiation source
- Filter/monochromator ( selector)
- Cells
- Detector
- Readout device
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.
Double beam instrument
Double-beam instrument with beams separated in space
• 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.
MOLECULAR SPECIES THAT
ABSORB UV/VISIBLE RADIATION
INORGANIC
SPECIES
ORGANIC
COMPOUNDS
CHARGE
TRANSFER
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
PERIODIC TABLE OF
ELEMENTS
• In UV/VIS spectroscopy, the transitions
which result in the absorption of EM
radiation in this region are transitions
between electronic energy levels.
ULTRAVIOLET-VISIBLE
SPECTROSCOPY
• In molecules, not only have electronic
level but also consists of vibrational and
rotational sub-levels.
• This result in band spectra.
Molecular absorption
Types of transitions
• 3 types of electronic transitions
- , and n electrons
- d and f electrons
- charge transfer electrons
H H O + + O H H O H H or
single covalent bonds (σ)
O C O or O C O
double bonds ()
N N N N
triple bond ()
or
What is σ, and n electrons?
lone pairs(n)
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.
• 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.
Pi () electron
• Examples of organic molecules containing
bonds.
CH2CH3
C
C
C
C
C
C
H
H
H
H
H
H
C HCCH3
ethylbenzene benzene
propyne
C
H
C
C
H
HC
HH
H
1,3-butadiene
• 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.
n electron
• Examples of organic molecules with non-
bonding electrons.
NH2
C
O
R
C C
H
HBr
H3C
:
.. :
..
.. : aminobenzene Carbonyl compound
2-bromopropene If R = H aldehyde
If R = CnHn ketone
• 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).
ABSORPTION BY ORGANIC COMPOUNDS
Electronic energy levels diagram
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Unoccupied levels
Occupied levels
n
*
*
Energ
y
*
*
n
*
n
*
Electronic transitions
*
*
n *
n *
In alkanes
In alkenes, carbonyl compounds, alkynes, azo
compounds
In oxygen, nitrogen, sulfur and halogen
compounds
In carbonyl compounds
Increasing
energy
* 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
Electronic 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
n * transitions
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
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
* 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
Examples n * and *
C C
O
HH
H
H
* at 180 nm
n * at 290 nm
(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)
MOLECULAR SPECIES THAT ABSORB
UV/VISIBLE RADIATION
• 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)
Absorption by organic compounds
CHROMOPHORES
Unsaturated organic functional
groups that absorb in the UV/VIS
region.
Absorption by organic compounds
Typical organic functional groups
that serve as chromophores Chromophores Chemical structure Type of transition
Acetylenic -CC- *
Amide -CONH2 *, n *
Carbonyl >C=O *, n *
Carboxylic acid -COOH *, n *
Ester -COOR *, n *
Nitro -NO2 *, n *
Olefin >C=C< *
• Groups such as –OH, -NH2 & halogens
that attached to the double bonded atoms
cause the normal chromophoric absorption
to occur at longer (red shift).
Absorption by organic compounds
AUXOCHROME
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.
• Factors that influenced the :
i) Solvent effects (shift to shorter : blue
shift)
ii) Structural details of the molecules
Absorption by organic compounds
Absorption spectra for typical organic
compounds
• Hypsochromic shift (blue shift)
- Absorption maximum shifted to shorter
• Bathochromic shift (red shift)
- Absorption maximum shifted to longer
Important terminologies
Nature of Shift Descriptive Term
To Longer Wavelength Bathochromic
To Shorter Wavelength Hypsochromic
To Greater Absorbance Hyperchromic
To Lower Absorbance Hypochromic
Terminology for Absorption Shifts
(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.
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
• 4f & 5f electrons
- Ions of lanthanide and actinide elements
- Their spectra consists of narrow, well-
defined characteristic absorption peaks.
Absorption by inorganic species
(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
Absorption spectra of aqueous charge transfer
complexes
• The fundamental law on which absorption
methods are based on Beer’s Law (Beer-
Lambert Law).
Quantitative Analysis
• 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.
Measuring Absorbance
• Calibration curve method
• Standard addition method
Quantitative Analysis
• 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.
Standard Calibration Curve
Ab
so
rban
ce
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.
How to produce standard calibration curve
• Prepare a series of
standard solution with
known concentration.
• Measure the absorbance of
the standard solutions.
• Plot the graph Abs vs
concentration of std.
• Find the “best’ straight line.
Stock solution
100 ppm
Calibration standard
Absorb
ance
• 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
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.
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.
Absorb
ance
Standard Addition Plot
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.
Standard Addition
Methods
Single-point standard
addition method Multiple standard
addition method
Standard addition
- if Beer’s Law is obeyed,
A = bVstdCstd + bVxCx
Vt Vt
= kVstdCstd + kVxCx
k is a constant equal to b
Vt
Standard Addition
- Plot a graph: A vs Vstd
A = mVstd + b
where the slope m and intercept b are:
m = kCstd ; b = kVxCx
• Cx can be obtained from the ratio of these
two quantities: m and b
b = kVxCx
m kCstd
Cx = bCstd
mVx
• 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.
Example:
Vol. of std added
(ml)
Absorbance
(A)
0 0.215
5.00 0.424
10.00 0.625
15.00 0.836
20.00 1.040
The results obtained:
Calculate the concentration of Fe3+ (in ppm)
in the raw-water sample
0
0.2
0.4
0.6
0.8
1
1.2
-10 -5 0 5 10 15 20 25
Ab
so
rban
ce
Vol. of std
Absorbance vs Vol. of std added
Slope, m = 0.0382
b = 0.24
(Vstd)0 = -6.31 ml
Note: From the graph, extrapolated value represents the volume of
reagent corresponding to zero instrument response.
• The unknown concentration of the analyte
in the solution is then calculated:
Csample = -(Vstd)0Cstd
Vsample
Cx = bCstd
mVx
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.
SELF-EXERCISE
Volume of
unknown (mL)
Volume of
standard (mL)
Absorbance
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.
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.
400 500 600 800 700
, nm
Violet 400-420
Indigo 420-440
Blue 440-490
Green 490-570
Yellow 570-585
Orange 585-620
Red 620-780
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”.
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.
Visible Spectroscopy
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!
lycopene, max = 474 nm
NH
HN
O
O
indigo
Visible Spectroscopy
