Fundamentals of Spectrophotometry

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 To Do:

1. Read Chapter 18 in Quantitative Chemical Analysis

Chapter 18: Fundamentals of Spectrophotometry

2. Listen to recorded lectures for Lessons 1-4.

3. Open and read ‘Reflection Calculation” (goes with topic 1).

4. Watch videos “Double Beam Spectrophotometer” (goes

with topic 3) and “How to Draw Jablonski Diagrams” (goes
with topic 5).

5. Complete Canvas quizzes

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 Topics

1. Properties of Light
2. Absorption of Light
3. Measuring Absorbance
4. Beer’s Law in Chemical Analysis
5. What Happens When a Molecule
Absorbs Light?
6. Luminescence

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 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). 4
 1. Properties of Light
Electromagnetic radiation (EMR) is a form of energy that is transmitted through
space at enormous velocities.

We often refer to EMR in the UV/visible and sometimes in the IR region as light.

EMR can be described as a wave with properties of wavelength, frequency, and

amplitude. Unlike sound waves, light can travel through vacuum – it does not
need a transmitting medium.

EMR is conveniently modeled as waves when dealing with phenomenon such as

reflection, refraction, interference, and diffraction.

The wave model fails to account for phenomena associated with the absorption
and emission of radiant energy.

For these, we must treat EMR as discreet packets of energy or particles known as
photons or quanta.
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 1. Properties of Light: Wave
Theory
• 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 6
In a medium containing matter, the velocity is slowed by interaction
between the electromagnetic field of the radiation and the electrons
of the matter.

Wavelength changes as
radiation passes from air into
dense glass and then back to
air. The wavelength shortens
by 200 nm, or more than 30%,
but the frequency of the
radiation is unchanged.

Wavenumber is defined as the number of waves per centimeter and

is equal to 1/λ; therefore units of cm-1. Used in IR spectroscopy.

The radiant power, P, in watts (W) is the energy of a beam that

reaches a given area per unit time.

Let’s look at some of the phenomenon observed when treating EMR

as waves. 7
A. Diffraction – the phenomenon
that occurs when a wave
encounters an obstacle (bending
of light) or a slit (spreading out of
light).

Thomas Young’s Experiment

Light from a single slit

produces coherent
light at the second
screen, where each
slit behaves as a
single source.

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If light exists as waves, the light waves will have interference under the
principle of superposition, creating bands of light (constructive
interference) and dark (destructive interference).

The appearance of the bands at a distant screen proved that light

exhibits both diffraction and interference.

The light that corresponds to direct transmission is called zero order (n =

0). The other maxima occur at angles represented by nonzero integers
n. n can be positive or negative, resulting in diffracted orders on both
sides of the zero order beam.
A diffraction grating is an optical
element that disperses white light into its
component wavelengths using a large
number of evenly spaced parallel slits.

Longer wavelengths (red) are diffracted

more than shorter wavelengths (blue).
Dispersion of light.
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B. Refraction – the change in direction of
a wave when it enters a medium where its
speed is different.
The refraction of light when it passes from
a fast medium to a slow medium bends
the light toward the normal boundary
between the two media.

The extent of sin 1 n2 v1

refraction is given  
by Snell’s Law: sin  2 n1 v 2

Violet light experiences a greater change in

velocity through the new medium than does
red light, and therefore greater difference
between the angle of incidence and the
angle of refraction.

Dispersion of light – separation into its

component wavelengths. 10
 Comparison of the spectra obtained from a
diffraction grating by diffraction (1), and a
prism by refraction (2). Longer wavelengths
(red) are diffracted more, but refracted less
than shorter wavelengths (violet).

http://en.wikipedia.org/wiki/Prism

C. Reflection – When radiation crosses an interface between media that

differ in refractive index, reflection always occurs.

The fraction of reflected radiation becomes greater with increasing

differences in RI.
Calculate the percent loss of intensity due to
reflection of a perpendicular beam of yellow light
I n  n  2
as it passes through a glass cell that contains

R 2 1 water. Assume that for yellow radiation the

I n  n  refractive index of glass is 1.50, of water is 1.33,

2

0 2 1 and of air is 1.00.

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D. Scattering – A very small fraction of radiation is transmitted in all
angles from the original path and the intensity of this scattered radiation
increases with particle size.

Rayleigh Scattering is the elastic scattering of light (at the same

wavelength, energy, and frequency as the incident photon) by particles
much smaller than the wavelength of light. This is the predominant mode
of scattering. The blue color of the sky is because of greater scattering of
the shorter wavelengths of the visible spectrum.

Raman Scattering it is also possible for the incident photons to interact

with molecules so that energy is either gained or lost and the scattered
photons are shifted in frequency – inelastic scattering.

For polarizable molecules, the incident photon can excite vibrational

modes of the molecules (molecules absorb energy from the photons), and
scattered photons are less in energy by the amount of the vibrational
transition energy.

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Spectra show lines below the Rayleigh scattering peak (at the incident frequency).
These are called “Stokes lines” and are said to be “red shifted”.

Molecules can also lose energy (give to the scattering photons). This results in a
scattered photon of higher energy and shifted to the blue side of the spectrum
compared to the incident beam (Rayleigh line). These lines are generally weaker
and are called “anti-Stokes lines”.

The difference in energy between the light from the original source and the
scattered light is equal to the difference in energy of vibrational levels in a molecule.

IR spectroscopy is not the only way information can be obtained on vibrational

transitions in a molecule. Raman spectroscopy is another method.

Spectrum showing Rayleigh and

Raman scattering and their relative
intensities.

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 1. Properties of Light: Particle
Theory
• The photoelectric effect is the observation that when light is shone onto
a piece of metal, a small current flows through the metal. The light is
giving its energy to electrons in the atoms of the metal and allowing
them to move around, producing the current. However, not all colors of
light affect metals in this way. No matter how bright a red light you have,
it will not produce a current in a metal, but even a very dim blue light will
result in a current flowing.

• If all else is equal, blue light has more energy than red light. If light is a
wave, a dim blue light would have the same amount of energy as a very
bright red light. And if this is the case, then why won't a bright red light
produce a current in a piece of metal as well as a dim blue light?

• Einstein realized that the only way to explain the photoelectric effect was
to say that instead of being a wave, light was actually made up of lots of
small packets of energy called photons that behaved like particles. 14
• 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?

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EMR Spectrum

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 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.
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• Transmittance is the
P P
fraction of original light T %T  100%
P0 P0
that passes through the
sample:
HW1 Q3-Q10

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

• Relating transmittance to P0
absorbance: A  logT log bc
P
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• 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.

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• 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.

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 Limitations to Beer’s Law
1. Real deviations – fundamental
limitations to the law. Beer’s law describes
the absorbance behavior of a medium
containing low concentrations of analyte. At
high analyte concentrations (>0.01M) there
are solute-solvent, solute-solute, and H-
bonding interactions.

2. Chemical deviations – the analyte

dissociates, associates, or reacts with
solvent to produce a product with a different
absorbance spectrum than the original
analyte specimen.
Figure shows the acid and base forms of
phenol red along with their UV spectra at
different pH.
HIn (color 1) ↔ H+ + In- (color 2)
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3. Instrumental deviations – result of
how absorbance measurements are made.
Always lead to negative absorbance
errors. Three of these defined below:

a) Polychromatic radiation – law assumes

that measurements are made with a source
of monochromatic radiation. But, we have
polychromatic sources followed by a
monochromator.

Important to choose
the wavelength of
maximum absorbance
(λmax) – maximum
response for a given
concentration of
analyte.

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b) Stray light – radiation exiting the Effect of stray light
monochromator is contaminated with
stray light from scattering/reflection off
of surfaces such as gratings, lenses,
mirrors, filters, and windows. Problem
because its wavelength often differs
greatly from what want to measure
AND because might not have even
passed through the sample.

Always limits the maximum absorbance

because when absorbance is high, the
radiant power transmitted through the
sample can become comparable to or
lower than the stray light level.

Spectrophotometers are most accurate when A = 0.4-0.9, if

absorbance is too high (too little light through) intensity is
too low to measure, if absorbance is too low (too much light
through) can’t distinguish between sample and reference.
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c) Mismatched cells – the cells holding the sample and the solvent blank
are not the same pathlength or do not have equivalent optical
characteristics.
A= ebc + k

Irreproducible positioning of the cuvet in the sample holder, dust on

containers or in samples (filter first), fingerprints on cuvet.

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 3. Measuring Absorbance
• Figure 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.

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• 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.

• Cuvets are typically made of fused-silica (SiO 2).

• 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.

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• 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. 27
• A double-beam spectrophotometer splits the light
alternately between sample and reference cuvets.
Watch video “Double Beam Spectrophotometer”
posted on Bb.

• 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.
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 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 29
pathlength. Find the concentration of benzene in mg/L.
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-

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• 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.
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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.

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• 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.

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 5. 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.
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• 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.
• Gives the 6 vibrations with the
corresponding wavenumbers in the
figure. 35
• 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 Watch video “How to
is called internal conversion Draw a Jablonski
Diagram” posted on Bb.
(IC).
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• 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
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heat.
• 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 to
10-10 s).

• Transition from T1 to S0
is called
phosphorescence
(lifetime is much
longer: 10-4 to 102 s).

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 6. 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.
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• 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.

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• 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.

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• In an emission
experiment, an excitation
wavelength λex is selected
and a luminescence is
observed 90o to the
incident light.
• An emission spectrum is a
graph of emission intensity
versus emission λem.
• An excitation spectrum is
a graph of the emission
intensity versus the
excitation λex.
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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.

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• 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:

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