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Lesson Objectives

Molecular UV-Vis Absorption
Spectroscopy
Jonyl L. Garcia, M.Sc., RCh.
Assistant Professor
Chemistry Department
At the end of this lesson, the student will be able to:
❑Understand the molecular-level theories of UV-Vis
absorption;
❑Differentiate between chromophores and
auxochromes;
❑Enumerate the steps in qualitative and quantitative
analysis using UV-Vis spectroscopy;
❑Perform calculations related to the applications of
UV-Vis spectroscopy.

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Molecular UV-Vis Absorption

SPECTROSCOPIC THEORIES ON
MOLECULAR ABSORPTION OF
UV-VIS RADIATION
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• Widely used for identification and determination of
myriad inorganic and organic species
• Generally results from the excitation of bonding
electrons
• Wavelengths of absorption peaks → correlated with the
types of bonds that exist in the species → useful as
SUPPORT DATA for structural elucidation.
• Useful for identifying functional groups in a molecule
• Three types of molecular species that ABSORB UV-Vis
radiation → (a) Organic compounds; (b) Inorganic
species; (c) Charge transfer complexes
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(a) Organic Species

• Absorption by
compounds that
contain:
– Shared electrons that
participate in bond
formation (σ, π
electrons)
– Unshared outer
electrons that are
largely localized about
such atoms as O, X, S
and N.

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Types of Transitions Types of Transitions

• σ-->σ* • n-->σ*
– Large ΔE: requires far – Lesser energy
UV radiation (not requirement than the
common)
σ→σ*.
– Shared electrons in
single bonds are firmly – Can be brought about by
held thus require high radiation between 150 –
energy excitation 250 nm (but still not very
– Absorption maxima < common) and are not
150 nm very intense
– This type of absorption – The compound must
corresponds to breaking contain atoms with
of σ bonds like C-C, C- unshared electron pairs,
H, C-O and C-X like O, S, N and X

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Types of Transitions Chromophores

• π*-->σ* • Functional groups that

– Symmetry forbidden absorb in the UV and Vis
• n→π*; π→π* regions (-C=C-, alkynes, -
– Most common electronic N=N-, -C=O-, -NO2-, -COO-,
transitions (UV to Vis): 200 -CONH-, -NO-, -ONO2-,
nm – 700 nm, very intense aromatic, etc)
– Exhibited by compounds
with unsaturated functional • High probability transition
groups → high molar absorptivity
– Electrons in unsaturated (103 – 105)
bonds are relatively loosely
held thus easily excited. • Medium probability
– A shift to higher λ is transition (ε = 102 – 103)
observed as the degree of
unsaturation increases • Low probability transition (ε
= 10-1 – 102)

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Effect of Auxochromes

• Auxochromes → Functional groups that do not

absorb in the UV/Vis region but have the effect of
shifting chromophore peaks to longer wavelengths

λmax
Benzene 256 nm
Aniline 280 nm
Phenol 270 nm

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Effect of Conjugation on Chromophores Solvent Effect

• Delocalized π electrons lowers the π* energy level • (a) in the gas phase, many lines
are seen due to electronic,
• This lowers the energy for the π→ π* transition thus vibrational and rotational
transitions; minimal interaction
shifts the wavelength of absorption to longer values between molecules
• (b) In a nonpolar solvent like
• Molecule absorbs over a wider region hexane, the electronic transitions
can be seen, but not the
– -C=C- vibrational and rotational
transitions due to some
– -C=C-C=C- interaction between solute and
– -C=C-C(=O)C=C- solvent molecules
• (c) In a polar solvent like water,
the strong intermolecular forces
have caused the electronic peaks
to blend resulting in a band
spectra

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Effect of Polar Solvents (b) Inorganic compounds

• Peaks associated with n→ π* transitions; shifted to shorter
wavelengths with increase solvent polarity due to increased • Most transition metal
solvation of unbonded electron pair which lowers the energy ions absorb in the UV-
of the n orbital. Vis region
• For π→ π* transitions, small red shift is observed with • Metals in the 1st and
increased solvent polarity due to attractive polarization forces 2nd transition metal
between the solvent and the absorber that tend to lower the
energy levels of both the unexcited and excited states. series contain filled
and unfilled d orbitals
• Bathochromic shift (red shift) → shift in λmax to higher that differ in energy as
wavelengths a consequence of
• Hypsochromic shift (blue shift) → shift in λmax to shorter splitting of energy
wavelengths levels when bonded to
ligands

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(b) Inorganic compounds (b) Inorganic compounds

• Lanthanide and actinide • Inorganic ions exhibit UV absorption peaks due to

elements also absorb UV/Vis
radiation as a result of n→π* transitions.
transitions by the 4f and 5f
electrons • Ex. Nitrates (313 nm), carbonates (217 nm), nitrites
• The electrons responsible for
absorption by these elements (360 nm and 280 nm), azides (230 nm) and
(4/ and 5f, respectively) are trithiocarbonate (500 nm) ions.
shielded from external
influences by electrons that
occupy orbitals with larger
principal quantum numbers.
As a result, the bands tend to
be narrow and relatively
unaffected by the species
bonded by the outer electrons

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(c) Charge transfer complexes

• These are exhibited by
inorganic and organic
complexes, which consists
of an electron donor group
bonded to an electron
acceptor
• Absorption of radiation
results from transfer of
electron from donor group
to an orbital associated
with the acceptor
• Show very large
absorptivities (> 10000),
hence many analytical
methods are based on this APPLICATIONS OF UV-VIS
type of complex
SPECTROPHOTOMETRY

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Qualitative Applications Qualitative Applications

• Sometimes useful for detecting the presence of
certain functional groups that act as chromophores
• The appearance of one or more peaks in the region
200 nm to 400 nm is a clear indication of the
presence of unsaturated groups or of atoms such as
S or halogens
• Identity of absorbing groups is evaluated by
comparing the spectrum of an analyte with those of
simple molecules containing various chromophoric
groups
• However, UV spectra lack sufficient fine structure
to lead to outright identification of analyte, UV
data must be supplemented with other data for
structural elucidation.

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Quantitative Applications Quantitation by UV-Vis Absorption

• UV-Vis absorption spectroscopy → one of the most

useful tools available to chemists for quantitative
analysis.
• Features
– Wide applicability
– High sensitivity
• Typical detection limits: 10-4 to 10-5 M but can go down to 10-6 to
10-7 M with procedural modification
– Moderate to high selectivity
– Good accuracy; relative errors ~ 1-5%
– Ease and convenience

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Considerations regarding solvents Glass vs. Quartz Cuvettes

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Quantitative Applications Quantitative Applications

• Applications to absorbing species • Application to non-absorbing species

– Organic compounds containing one or more chromophoric
groups and inorganic species that absorb UV-Vis radiation
can be determined by direct measurement of
absorbance/transmittance
– Non-absorbing inorganic and organic analytes can be
reacted with numerous selective reagents to yield
products that absorb strongly in the UV-Vis regions.
– Requires that the color forming reaction goes near
completion
– Color forming reagents can also be employed for
determining already absorbing species
• E.g. transition metal ions; the molar absorptivity of the product
will be frequently orders of magnitude greater than the original
uncombined species.

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Use of Chelating Agents

PROCEDURAL DETAILS FOR

QUANTITATIVE ANALYSIS

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(1)Selection of Wavelength (2)Variables that Influence Absorbance

• Absorbance measurements • Common variables that

are made at a wavelength
corresponding to the λmax influence absorbance include:
– Leads to maximum – Nature of solvent
sensitivity
– Absorption curve is often flat – pH of solution
at this region → leads to
good adherence to Beer’s – Temperature
law
– Measurements are less – High electrolyte concentration
sensitive to uncertainties
arising from failure to
– Presence of interfering
reproduce precisely the substances
wavelength setting of the
instrument • The effect of these variables
must be optimized.

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(3)Determination of Relationship between

Absorbance and Concentration
(4)Standard Addition Method

• Necessary to prepare a calibration curve from a
series of standard solutions; standards should
approximate the overall composition of the actual
samples and cover reasonable concentration range
of analyte
• Prepare at least 6 standards, whose concentration
encompasses the expected concentration range of
the sample → Method of External Calibration
• A method used to counter the matrix effects arising
from the difference in overall composition between
the sample and an external standard.
• Involves adding one or more increments of a
standard solution to sample aliquots of the same
size, each solution is then diluted to a fixed volume
prior to absorbance measurements.

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(4)Standard Addition Method

Multiple Standard Addition Two-Point Standard Addition

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(5)Analysis of Mixtures of Absorbing

Substances
• The total absorbance
of a solution at a given
wavelength is the sum
of the absorbances of
the individual
components present
• This relationship makes
possible the
quantitative
determination of the
individual constituents
of a mixture, even if
their spectra overlap. A = A1 + A2 + ……… An

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(5)Analysis of Mixtures of Absorbing

Substances
• The molar absorptivities can be
evaluated using the individual
standard solutions
• A1 and A2 can be determined
experimentally; b is also a given
variable (cell thickness) At λ2
• cN and cM can be computed using the
two solutions (How?)
• Valid only if Beer’s law is obeyed and
if the two components behave
independently of each other
• Greater accuracy obtained if At λ1
wavelengths at which the
differences in molar absorptivities
are selected
• Mixtures containing more than two
absorbing species can be analyzed in
principle.

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(6)Photometric titrations (6)Photometric titrations

• Can be used in locating • Photometric titration

the equivalence point curve is a plot of
of a titration, provided absorbance, corrected
the analyte, the for volume changes, as a
reagent, or the function of volume of
titration product titrant
absorbs radiation. • Consist of 2 straight line
regions with differing
• Alternatively, an slopes
absorption indicator • End point is the
can provide the intersection of the
absorbance necessary extrapolated linear
for location of portions of these 2 line
equivalence segments

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(7) Spectrophotometric Studies of Complex

(6)Photometric titrations
• Advantages of Photometric Titration
– Often provided more accurate results than direct
photometric analysis
– Presence of other absorbing species may not interfere
because only change in absorbance is being measured
– Reactions need not have such favorable equilibrium
constants
– Dilute solutions may be titrated.
• Application of photometric titrations
– Applied to all type of titrations: neutralization,
oxidation/reduction, complexation, precipitation
Ions
• Spectrophotometry is a valuable tool for
determining the composition of complex ions in
solution and for determining their formation
constants.
• The composition of a complex in solution can be
determined without actually isolating the complex
as a pure compound.
• The power of the technique lies in the fact that
quantitative absorption measurements can be
performed without disturbing the equilibria under
consideration.

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(7) Spectrophotometric Studies of Complex

Ions
(7a) Method of Continuous Variations

• The three most common techniques used for • Cation and ligand
complex-ion studies are: solutions with identical
analytical concentrations
– (a) the method of continuous variations are mixed in such a way
– (b) the mole-ratio method that the total volume
– (c) the slope-ratiomethod. and the total moles of
reactants in each
mixture are constant but
the mole ratio of
reactants varies
systematically
• E.g. 1:9, 8:2, 7:3, and so
forth

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(7a) Method of Continuous Variations (7a) Method of Continuous Variations

• The absorbance of each • The curvature of the

solution is then measured experimental lines is the result
at a suitable wavelength of incompleteness of the
and corrected for any complex-formation reaction.
absorbance the mixture • A formation constant for the
might exhibit if no reaction complex can be evaluated from
had occurred. measurements of the
• The corrected absorbance deviations from the theoretical
is plotted against the straight lines, which represent
volume fraction of one the curve that would result if
reactant, that is, VM /(VM + the reaction between the
VL), where VM is the volume ligand and the metal
of the cation solution and proceeded to completion.
VL is the volume of the
ligand solution.

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(7b) Mole-Ratio Method (7b) Mole-Ratio Method

• a series of solutions is • If the formation constant

prepared in which the is reasonably favorable,
two straight lines of
analytical different slopes that
concentration of one intersect at a mole ratio
reactant (usually the that corresponds to the
metal ion) is held combining ratio in the
constant while that of complex are obtained
the other is varied. • Formation constants can
be evaluated from the
• A plot of absorbance data in the curved
versus mole ratio of portion of mole-ratio
the reactants is then plots where the reaction
is least complete.
prepared.

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(7b) Mole-Ratio Method (7c) Slope-Ratio Method

• A mole-ratio plot may • Particularly useful for weak complexes but is

reveal the stepwise applicable only to systems in which a single complex
formation of two or
more complexes as is formed.
successive slope • Assumptions:
changes if the
complexes have – the complex-formation reaction can be forced to
different molar completion by a large excess of either reactant,
absorptivities and their – that Beer’s law is followed under these circumstances,
formation constants
are sufficiently – That only the complex absorbs at the wavelength chosen.
different from each
other.

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Instrumental Indeterminate Errors in

(7c) Slope-Ratio Method Transmittance Measurements
𝜺𝒃𝒄𝑴ൗ
𝑨𝟏 = 𝜺𝒃 𝑴𝒙 𝑳𝒚 = 𝒙
𝜺𝒃𝒄𝑳ൗ
𝑨𝟐 = 𝜺𝒃 𝑴𝒙 𝑳𝒚 = 𝒚

𝜺𝒃ൗ 𝒚
𝒙൘
𝜺𝒃ൗ = 𝒙
𝒚

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THANK YOU FOR YOUR

ATTENTION!

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