Chapter 18

Fundamentals of Spectrophotometry
Colorimetric Dubois, M., Gilles, 1956 Analytical 39594
Method for K.A., Hamilton, Chemistry28(3),
Determination of J.K., Rebers, pp. 350-356
Sugars and Related P.A., Smith, F.
Substances
 Overview

18-1 Properties of Light

18-2 Absorption of Light
18-3 Measuring Absorbance
18-4 Beer’s Law in Chemical Analysis
18-5 Spectrophotometric Titrations
18-6 What Happens When a Molecule Absorbs
Light?
18-7 Luminescence
Spectrometry and Spectroscopy are two terms which are very commonly
confused. Both are types of techniques that we use to characterise molecules but
they work in very different ways.

Spectroscopy
Spectroscopy is the study of how materials respond to electromagnetic radiation
– that is, radiation with a wavelength from the electromagnetic spectrum.
Examples include:
Ultra-Violet visible (UV-vis) adsorption spectroscopy; studies molecules
which absorb UV-visible radiation. This is absorbed by valance electrons in a
molecule.
Infrared (IR) absorption spectroscopy; studies molecules which absorb IR
radiation. This can result in the bending and stretching of atomic bonds. This can
help us to work out what functional groups are in a molecule.
Nuclear Magnetic Resonance (NMR) spectroscopy; studies how molecules
respond to radio frequencies. In particular this causes some atomic nuclei to
absorb energy and helps us to understand the environment they are in.

Spectrometry
Mass Spectrometry does not involve exposing a molecule to radiation. Instead it
relies on high energy particles (such as electrons) to charge and fragment (break
up) a molecule. The charged species are then separated according to their mass
to charge ratio (m/z).
 Fundamentals of Spectrophotometry
• Spectroscopy is the theoretical approach to the
science of studying the interaction between matter
and radiated energy.
• Spectrometry is the practical application of
spectroscopy. Spectrometry uses instruments called
spectrometers.
• Spectrophotometry is the method used to measure
how much a chemical substance absorbs light as a
beam of light passes through a sample solution.
• Spectrophotometers are the instruments used to
quantitatively measure the reflection or transmission
properties of a material as a function of wavelength
(a spectrum).
 18-1: Properties of Light
• Wavelength (λ) is the linear distance between
successive maxima or minima.
• Frequency (ν) is the number of oscillations of the
field that occur per second; has units of sec-1 (Hz).

• The product of frequency times wavelength is equal

to the speed of light, c, which has a value of 3.0 x
108 m/s: νλ = c/n
• (n= refractive index of the medium, equals 1 for
vacuum)
 18-1: Properties of Light
• Photons are particles of light with energy:

E = hν h = Planck’s constant = 6.626 x 10-34 J-s

• Combining the two equations:

Example: By how many kilojoules per mole is the

energy of O2 increased when it absorbs ultraviolet
radiation with a wavelength of 147 nm?
18-1: Properties of Light
 18-2: Absorption of Light
• The energy of a molecule
increases when it absorbs
a photon.
• When light is absorbed by
the analyte, the irradiance
(the energy per second
per unit area of the light
beam), P, decreases.
• When monochromatic light
(consists of one
wavelength) with
irradiance P0 strikes the
sample, the irradiance of
the beam emerging is P,
where P≤P0.
 18-2: Absorption of Light
• Transmittance is the P P
fraction of original light T %T   100%
P0 P0
that passes through the
sample:

• Absorbance, A, of
solutions contained in a
transparent cell having a Beer’s law: A = εbc
pathlength of “b” cm:

• Relating transmittance to P0
A  - logT  log  bc
absorbance: P
 18-2: Absorption of Light
• Absorbance is proportional to the concentration of
the light-absorbing molecules in the sample.

• The part of the molecule responsible for light

absorption is called a chromophore.

Example: Find the absorbance and transmittance of a

0.00240 M solution of a substance with a molar
absorptivity of 313 M-1cm-1 in a cell with a 2.00 cm
pathlength.
 18-2: Absorption of Light
• Beer’s law is a limiting law:

- It applies to monochromatic light.

- It applies to dilute solutions ≤0.01 M.
- It fails if the analyte undergoes a chemical change
and the product of this change has a different
spectrum than the original analyte.

Beer’s law fails when deviations from the

proportionality between measured absorbance and
concentration occur.
 18-3: Measuring Absorbance
• Figure 18-5 shows the minimum requirements for a
spectrophotometer.

• Light from the continuous source passes through a

monochromator, which selects a narrow band of wavelengths from
the incident beam.

• This light passes through the sample of pathlength b, and the

irradiance of the emergent light is measured.

• For visible and ultraviolet, a liquid sample is usually contained in a

cell called a cuvet.
 18-3: Measuring Absorbance
• Cuvets are typically made of fused-silica (SiO2).
• Glass is OK for visible but not for ultraviolet because it
absorbs UV radiation.
• The most common pathlength is 1.000 cm, sold in
matched sets for sample and reference.
 18-3: Measuring Absorbance
• For IR, cells are constructed of NaCl or NaBr.
• Solids samples are ground into a fine powder,
which can be added to mineral oil (Nujol) to give a
dispersion called a mull that is pressed between
two KBr plates.
• A single-beam spectrophotometer has only one
beam of light.
• We measure the irradiance of light passing through
a reference blank, P0.
• The cuvet is removed and replaced by an identical
one containing the sample.
• The irradiance of light passing through the sample,
P, is measured.
 18-3: Measuring Absorbance
• A double-beam spectrophotometer splits the light
alternately between sample and reference cuvets.

• When recording an absorbance spectrum, first

record a baseline spectrum – this will be
subtracted from the sample’s absorbance.

• Choose the wavelength of maximum absorbance

because:
1.The analysis has the greatest sensitivity.
2.There is little variation in the absorbance versus
wavelength.
 18-3: Measuring Absorbance
• Absorbance should be ~0.4-0.9.
- Too much light absorbed makes the intensity hard to
measure.
- Too little light absorbed makes it hard to distinguish
between the blank and the sample.

• The less noise, the

lower the concentration
of analyte that can be
detected.
• Place cuvets into the
instrument reproducibly
and wipe off fingerprints
and dust.
 18-4: Beer’s Law in Chemical Analysis
• For a compound to be analyzed by spectrophotometry, it
must absorb light, and this absorption should be
distinguishable from that due to other substances in the
sample.

Example: a) Pure hexane has a negligible ultraviolet

absorbance above a wavelength of 256 nm. A solution
prepared by dissolving 25.8 mg of benzene (MM 78.11) in
hexane and diluting to 250.0 mL had an absorption peak at 256
nm and an absorbance of 0.266 in a 1.000-cm cell. Find the
molar absorptivity of benzene at this wavelength.

b) A sample of hexane contaminated with benzene has an

absorbance of 0.070 at 256 nm in a cuvet with a 5.000-cm
pathlength. Find the concentration of benzene in mg/L.
 18-4: Beer’s Law in Chemical Analysis
Example: Serum Iron Determination has three steps:

1. Reduce Fe3+ in transferrin to Fe2+.

2. Add trichloroacetic acid to precipitate proteins, leaving

Fe2+ in solution. Centrifuge the mixture to remove the
precipitate.

3. Transfer a measured volume of supernatant liquid

from Step 2 to a fresh vessel. Add buffer plus excess
ferrozine to form a purple complex.
The absorbance is measured at 562 nm
Fe2+ + 3 ferrozine2- → (ferrozine)3Fe4-
18-4: Beer’s Law in Chemical Analysis
• Serum copper also forms
a colored complex with
ferrozine.

• Interference is eliminated
by adding neocuproine or
thiourea.

• These reagents mask

Cu+ by forming strong
complexes that prevent
Cu+ from reacting with
ferrozine.
 18-4: Beer’s Law in Chemical Analysis
Example: Serum iron and standard iron solutions were analyzed
as follows:

Step 1: To 1.00 mL of sample, add 2.00 mL of reducing agent

and 2.00 mL of acid to reduce and release Fe from transferrin.
Step 2: Precipitate proteins with 1.00 mL of 30 wt%
trichloroacetic acid. Centrifuge the mixture to remove protein.
Step 3: Transfer 4.00 mL of supernatant liquid to a fresh tube and
add 1.00 mL of solution containing ferrozine and buffer. Measure
the absorbance after 10 min.
Step 4: To establish each point on the calibration curve in the
figure, use 1.00 mL of standard containing 2-9 mg Fe in place of
serum.
 18-4: Beer’s Law in Chemical Analysis
• The blank absorbance was
0.038 at 562 nm in a 1.000-cm
cell.
• A serum sample had an
absorbance of 0.129. The
blank was subtracted from each
standard absorbance.
• The least-squares line through
the standard points is:
Absorbance = 0.0670 x (mg Fe in the
sample) + 0.0015

• Find the concentration of iron in

the serum.
18-4: Beer’s Law in Chemical Analysis
 18-5: Spectrophotometric Titrations
• Monitor changes in absorbance during a titration to
determine when the equivalence point has been
reached.
• A solution of the iron-transport protein, transferrin, can
be titrated against iron to measure the transferrin
content; each transferrin molecule binds to two Fe 3+
ions.
• Transferrin without iron is colorless and called
apotransferrin.
• When Fe3+ binds to the protein, a red color with an
absorbance maximum at a wavelength of 465 nm
develops:

Apotransferrin (colorless) + 2Fe3+ → (Fe3+)2transferrin (red)

 18-5: Spectrophotometric Titrations
• The titration of 2.000 mL of
apotransferrin with 1.79 mM
ferric nitrilotriacetate is shown in
Figure 18-11.
• As iron is added to the protein,
red color develops and the
absorbance increases.
• When the protein is saturated
with iron, no more can bind and
the curve levels off.
• The extrapolation of the titration
curve at 203 mL in the figure is
taken as the end point.
 18-5: Spectrophotometric Titrations
• Ferric nitrilotriacetate absorbs at 465 nm; therefore
absorbance increases slowly after the equivalence
point.

• The quantity of Fe3+ required for complete reaction in

the figure is (203 x 10-6 L) x (1.79 x 10-3 mol/L) =
0.363 mmol, and the moles of protein in the sample
must be half of that (0.182 mmol) since two Fe3+ bind
per mole of protein.

• Dilution must be considered in making the plot

because the volume is different at each point.
 18-6: What Happens When a Molecule
Absorbs Light?
• When a molecule
absorbs a photon, it is
promoted to an
excited state.
• When a molecule
emits a photon, the
energy of the
molecule falls by an
amount equal to the
energy of the photon.
 18-6: What Happens When a Molecule
Absorbs Light?
• Infrared and microwave radiation are not
energetic enough to induce electronic
transitions, but they can change the
vibrational or rotational motion of a molecule.
• For the four atoms and three
axes in space, formaldehyde
has a total of 12 ways it can
move.
• Three of these are
translational motion in the x,
y, and z directions.
• Three of these are rotations
about the x, y, and z axes of
the molecule.
 18-6: What Happens When a Molecule
Absorbs Light?
• With promotion from S0 to a
vibrationally and rotationally
excited level of the S1, usually
the first thing that happens is
vibrational relaxation to the
lowest vibrational level of S1.
• This is a nonradiative transition
(labeled R) because no photon
is emitted.
• From S1, a molecule can enter
a highly excited vibrational
level of S0 having the same
energy as S1. This is called
internal conversion (IC).
 18-6: What Happens When a Molecule
Absorbs Light?
• In the path A-R1-IC-R2, the
entire energy of the photon will
have been converted into heat.
• From S1, a molecule could
cross into an excited vibrational
level of T1, called intersystem
crossing (ISC).
• After radiationless R3, the
molecule is in the lowest
vibrational level of T1 and could
undergo a second intersystem
crossing to S0, followed by R4;
all energy of the photon is
converted to heat.
18-6: What Happens When a Molecule
Absorbs Light?
• A molecule could relax from
S1 or T1 to S0 by emitting a
photon.

• Transition from S1 to S0 is
called fluorescence (lifetime
is short 10-8 to10-10 s).

• Transition from T1 to S0 is
called phosphorescence
(lifetime is much longer: 10-4
to 102s).
 18-7: Luminescence
• Fluorescence and
phosphorescence are
examples of luminescence –
the emission of light from an
excited state molecule.
• Luminescence is more
sensitive than absorption
and can observe single
molecules.
• The emission spectrum is
roughly the mirror image of
the absorbance spectrum.
18-7: Luminescence
• Fluorescence and
phosphorescence come at a
lower energy (longer λ) than
absorption.
• After absorption, the vibrationally
excited S1 molecule relaxes
back to the lowest vibrational
level of S1 before emitting a
photon.
• Emission from S1 can go to any
vibrational level of S0.
• The highest energy transition is
λ0, with a series of peaks
following at longer λ.
18-7: Luminescence
• An excitation spectrum is
nearly the same as an
absorbance spectrum.

• In emission spectroscopy
we measure the emitted
radiation, rather than the
fraction of incident
radiation striking the
detector.
18-7: Luminescence
 18-7: Luminescence
• Luminescence intensity is measured by:

where P0 is the irradiance incident on the sample cell, which is

absorbed over pathlength b1.

• The irradiance of the beam when it has traveled the additional

distance b2 is:

• Emission intensity I is proportional to

irradiance absorbed:

where k' is the proportionality constant.

 18-7: Luminescence
• Emission intensity is given by Beer’s law:

• The relationship between incident irradiance and

emission intensity is:

• The emission intensity at low concentration is:

 18-7: Luminescence
Example: Fluorimetric Assay of Selenium in Brazil Nuts
• To measure selenium in Brazil nuts, 0.1 g of nut is
digested with 2.5 mL of 70 wt% HNO3.
• H2SeO4 in the digest is reduced to H2SeO3 (selenite),
which is then derivatized to form a fluorescent product
that is extracted into cyclohexane.

• Maximum response of the fluorescent product was

observed with an excitation wavelength of 378 nm and
an emission wavelength of 518 nm.
 18-7: Luminescence
• Some analytes such as riboflavin and polycyclic
aromatic compounds are naturally fluorescent.
• A fluorescent moiety, such as fluroescein, can be
coupled to other molecules in order to form derivatives
that fluoresce.

• Chemiluminescence is the
emission of light from a chemical reaction: