CHE 312_LECTURE 3

Chemical analysis using a

spectrophotometers

Single-beam spectrophotometer- uses only one

beam in which the sample and reference cuvettes
are alternately placed in the beam path
Double-beam spectrophotometer- uses two
beams where one beam is passed through the
sample and the other beam is passed through the
reference
Block diagrams for both a single-beam
and double-beam spectrophotometer
 Single-beam spectrophotometers
Uses only one beam, the sample and reference are
alternately placed in the beam (i.e in the sample
slot) at each wavelength to be measured.
 If the radiant power of the radiation emerging
from the sample is P and that emerging from the
reference is Po then A=log Po -log P
 The purpose of comparing the sample to the
reference solution compensate for reflection,
scattering and absorption by both the cuvette and
the solvent
 Disadvantages of single-beam
spectrophotometers
The inconvenience of alternately removing the
reference to place the sample cuvette for each
wavelength being measured
Not suitable for measuring absorbance as a
function of time especially in kinetic
experiments
 This is because both the beam intensity and the
detector response may drift slightly with time
 Double-beam spectrophotometers
It uses two beams in which one beam is passed
through the sample and the second one through
the reference solution
 This is achieved by the use of a mirror rotated by a
motor to direct the beam alternately to sample
and reference cuvettes
 The radiant powers P and Po are compared: A=log
Po /P
 Advantages of double-beam
spectrophotometer
Automatic correction for any changes in beam
intensity and detector response
Automatic wavelength scanning and continuous
recording of absorbance
 Chemical analysis
In spectrophotometric analysis, the procedure
involves;
 First obtaining a scan (spectrum) of the pure
solvent or reagent blank or reference in the
expected wavelength range
 Then the sample is scanned and hence True
absorbance=Sample absorbance-blank
absorbance
 The absorbance readings are done at the
wavelength of maximum absorbance (λmax)
NB: Why are absorbance readings taken at
wavelength of maximum absorbance?
Absorbance is constant at λmax because there is
minimum drift in detector response/source
intensity
For a given concentration, ε is maximum at λmax
Because Amax=εmax bc
Most spectrophotometers exhibit their minimum
relative uncertainty (i.e less drift) in the range
A=0.4-0.9 and this because;
 If P<< Po, i.e high absorbance, A>0.9
 If P is almost equal to Po, i.e low absorbance,
hence it is difficult to distinguish between the
sample and the reference for very low
absorbance values
Precautions
Prevent stray light from entering the detector-the
sample compartment must be light-tight
 Stray light will interfere with absorbance
measurements
Prevent dust from entering the beam path to
avoid scattering of light
Fingerprints on the faces of silica cuvettes absorb
radiation-handle the cuvettes with tissue paper
Avoid mismatch of the sample and reference
cuvettes since this will lead to systematic error in
comparing P and Po
Avoid misplacement of the cuvette in the sample
compartment as this will lead to random error
 Principles of Uv/Vis
Absorption of radiation in the visible(Vis) and
ultraviolet(UV) regions of the electromagnetic
spectrum results in electronic transitions
between molecular orbitals.
The UV range extends from 100-400 nm of which
100-190 nm is known as far UV and 190-400 nm
is called near UV
Visible range extends from 400-800 nm
Commercial UV/Vis instruments cover 200-1000
nm region
 Nature of Electronic Transitions
 When two atoms react to form a compound,
electrons from both atoms participate in the
bond and occupy a new molecular orbital
where bonding electrons are associated with
the molecule as a whole
This is called a bonding orbital and represents
the lowest energy level
Simultaneously, a corresponding antibonding
obital is also formed, which are vacant in the
unexcited or groundstate
Thus a covalent bond may be formed by
combination of S orbitals or P orbitals. This are
designated as σ and π bonds respectively
Corresponding antibonding orbitals are
designated as σ* and π*
Valence electrons NOT involved in bonding are
referred to as non-bonding electrons and
designated n
In organic molecules, n electrons (lone pairs)are
located in the atomic orbitals of nitrogen,
oxygen, sulphur and halogen atoms regions
include
The possible electronic transitions involved in
uv/vis include σ → σ*, n → σ*, n → π* and π →
π*
And their relative energies in decreasing order
σ → σ* > n → σ* > π → π*>n → π*
See Figures in the next two slides which depict
the relative energies of the possible electronic
transitions
• σ → σ* and n → σ* transitions -These are high-
energy transitions and involve very short
wavelength ultraviolet light (< 150 nm) e.g
observed for alkanes.
• These transitions are NOT useful analytically as
they fall outside the generally available
measurable range of UV-visible
spectrophotometers (200-1000 nm)
• π →π* and n → π* transitions- these are low-
energy transitions and fall within measurable
range of UV-visible spectrophotometers (200-
1000 nm)
The most intense band (large ε) for compounds is
mostly due to π →π* transition.
The n → π* transition is of lowest energy but is of
low intensity (weak absorption or low ε ) as it is
symmetry forbidden.
 Why Broad bands In UV/Vis Spectra
The transition of an electron from one energy
level to another is accompanied by
simultaneous change in vibrational and
rotational states and causes transitions
between various vibrational and rotational
levels of lower and higher energy electronic
states
Therefore many radiations of closely placed
frequencies are absorbed and a broad
absorption band is obtained (see caffeine
Absorption spectrum in the next slide)
The most probable transition would appear to
involve the promotion of one electron from the
highest occupied molecular orbital (HOMO) to the
lowest unoccupied molecular orbital (LUMO), but
in many cases several transitions can be observed,
giving several absorption bands in the spectrum
 important terms and definitions
Chromophore: The group of atoms within a
molecule containing electrons responsible for
the absorption of radiation that causes
electronic excitation.
Examples –
C=C, C=O, C=S, N=N, N=O
Auxochrome: The substituents that themselves do not
absorb ultraviolet radiations but their presence shifts the
absorption maximum to longer wavelength (red shift)
with a corresponding increase in absorption intensity.
 Some examples are substituents like, hydroxyl, alkoxy,
halogen, amino group etc.
All auxochromes have one or more non-bonding pairs of
electrons. If an auxochromes is attached to a
chromophore, it helps is extending the conjugation by
sharing of non-bonding pair of electrons
Bathochromic Shift or Red shift: A shift of an absorption
maximum towards longer wavelength or lower energy.
Hypsochromic Shift or Blue Shift: A shift of an absorption
maximum towards shorter wavelength or higher energy
 Factors affecting absorption by a
chromophore
Solvents Effects
 Absorption bands arising from n → π* transitions
suffer hypsochromic shifts on increasing the solvent
polarity, whilst those of π → π* transitions are
shifted bathochromically
 Explanations lie in the fact that the energy of the
non-bonding orbital n is lowered by hydrogen
bonding in the more polar solvent thus increasing the
energy of the n → π* transition but the energy of the
π* orbital is decreased relative to the n orbital
 Non-polar vs polar solvents
• There is little or no effect on the uv/vis absorption
spectrum of an analyte dissolved in a non-polar
solvent
• However, If the SAME analyte dissolved in a polar
solvent , the absorption bands are broadened,
which leads to a loss in structural resolution and
reduction in εmax.
• The absorption spectrum of phenol (analyte) in
isooctane (non-polar solvent) and ethanol (polar
solvent) is illustrating this effect (see next slide)
Spectra of phenol in isooctane and
ethanol
  Conjugation Effects
 Absorption bands due to conjugated
chromophores are shifted to longer wavelengths
(bathochromic or red shift) and intensified (large
ε )relative to an isolated chromophore
 The shift can be explained in terms of interaction
or delocalization of the π and π* orbitals of each
chromophore to produce new orbitals in which
the highest π orbital and the lowest π* orbital
are closer in energy
e.g Effect of conjugation on absorption of
ethylene
The longer the conjugated carbon chain in the
absorbing system, the greater the intensity of
the absorption( large ε) .
This is shown by the spectra of the polyenes CH3-
(CH=CH)n-CH3, where n=3,4 and 5 (next slide).
• β-carotene, a vitamin found in carrots, and used in
food colouring, has eleven conjugated double
bonds (Fig below) and its absorption maximum has
shifted out of the ultraviolet and into the blue
region of the visible spectrum, hence it appears
bright orange
Some Uv/Vis Analytes (find more examples
of your own)
Nicotine
 Practice questions 2
1. Discuss limitations to Beers law in quantitative
analysis
2. With the aid/help of a labeled block/schematic
diagrams, describe how a) a single-beam
spectrophotometer b) double-beam
spectrophotometer works.
3. Define the following terms as applied to
spectroscopy; molar absorptivity, spectrometry,
Absorption spectrum, transmittance
4. Pure hexane has negligible ultraviolet absorbance
above a wavelength of 200 nm. A solution prepared
by dissolving 25.6 mg of benzene (C6H6,
MM=78.114 g/mol) in hexane and diluting to 250.0
ml had an absorption peak at 256 nm and an
absorbance of 0.266 in a 1.00 cm cell. Find the
molar absorptivity of benzene at this wavelength
(256 nm)
5. Describe the changes in matter when it interacts
with: X-rays, UV/Vis, IR, microwave and
radiofrequency electromagnetic radiation
6. Calculate the frequency in hertz of;
i. An emission line of copper at 324.7 nm
ii. An X-ray beam with a wavelength of 2.97 Å

7. Calculate the wavelength in nm of

iii. An airport tower transmitting at 118.6 MHz
iv. An infra-red absorption peak with a
wavenumber of 1375 cm-1
8. Calculate the frequency in hertz and the
energy in joules of an X-ray photon with a
wavelength of 2.35 Å
9. Calculate the wavelength and the energy in
joules associated with a signal at 220 MHz
10. Express the following absorbances in terms
of percent transmittance
i. O.936
ii. 0.494
iii. 0.0350
10. Convert the accompanying transmittance
data to absorbances;
i. 22.7 %
ii. 31.5 %
iii. 0.103
iv. 0.567
11. The complex formed between Cu(I) and 1,10-
phenanthroline has a molar absorptivity of 7000 L cm-1
mol-1 at 435 nm, the wavelength of absorption. Calculate
i. The absorbance of a 6.77 x 10-5 M solution of the
complex when measured in a 1.00 cm cell(cuvet) at
435 nm.
ii. The percent transmittance of the solution in (i)
iii. The concentration of a solution in a 5.00 cm cell that
has the same absorbance as the solution in (i)
iv. The path length through a 3.40 x 10-5 M solution of
the complex that is needed for an absorbance that is
the same as the solution in (i)