Module contents

Atomic Molecular
Electrochemical
spectrometry spectrometry
• AAS • UV-vis • ISE
• ICP-AES • IR
• MS

Separation Others

• Chromatography • Statistics
• Lab management

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 Question

• Benzene is a common impurity in

cyclohexane.
• How do you measure its concentration?

2
 Department of Chemical and
Environmental Engineering
CHEE 2023 Analytical Measurement

Lecture 2:
UV-Visible Spectroscopy

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 Learning Outcomes (LOs)
1. Understand the principles and applications of atomic and
molecular spectroscopy, then compare and contrast them.

2. Define and identify the types of errors that can arise from
measurements, apply statistical equations to analyse these
errors, then suggest ways to overcome them.

3. Demonstrate the ability to calibrate, process and interpret results

from analytical instruments.

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 Overview

• Review: the electromagnetic spectrum

• Absorption theory
• The spectrometer
• The Beer-Lambert Law
• Quantification procedures
• Applications
• Questions.

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Review: The Electromagnetic spectrum
 Introduction
• Visible light absorption is known to all of us, because this is what causes objects to be coloured.
• E.g. a blue dye appears blue because the light at the red end of the spectrum is absorbed, leaving the
blue light to be transmitted (refer to diagram).

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 Regions of the electromagnetic spectrum

Region Ultraviolet Visible Infrared

Wavelength (nm) 185 – 400 400 – 700 700 – 1100
Typical energy (eV) 4 1 0.1

When light is absorbed by a material, valence electrons are promoted from their normal
(ground) states to higher energy (excited) states. 8
 UV-Visible Spectrometer
• Because only small numbers of absorbing molecules are required, it is convenient to
have the sample in solution

ideally the solvent should not absorb in the ultraviolet/visible range however, this is rarely
the case.

• In conventional spectrometers electromagnetic radiation is passed through the

sample which is held in a small square-section cell (usually 1 cm wide internally).

• Radiation across the whole of the ultraviolet/visible range is scanned over a period of
approximately 30 s

and radiation of the same frequency and intensity is simultaneously passed through a
reference cell containing only the solvent.

• Photocells then detect the radiation transmitted and the spectrometer records the
absorption by comparing the difference between the intensity of the radiation passing
through the sample and the reference cells (Fig. next page).

• In the latest spectrometers radiation across the whole range is monitored

simultaneously.

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 UV-Visible Spectrometer (2)
Double-beam:

• Measures the absorbance of UV or visible light by a sample, either at:

a single wavelength, or,

scan over a range in the spectrum.

• UV region: 185 to 400 nm; visible region: 400 to 700 nm.

• Used both quantitatively and qualitatively.

• Light source

a combination of tungsten/halogen and deuterium lamps.

The output from the light source is focused onto the diffraction grating which splits the incoming light into its
component colours of different wavelengths, like a prism (shown) but more efficiently.
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 UV-Visible Spectrometer (3)
• No single lamp provides radiation across the whole of the range
required, so two are used.

A hydrogen or deuterium discharge lamp covers the ultraviolet range,
and a tungsten filament (usually a tungsten/halogen lamp) covers the
visible range.

• The radiation is separated according to its frequency/wavelength by

a diffraction grating followed by a narrow slit.

• The slit ensures that the radiation is of a very narrow waveband –

i.e. it is monochromatic.

• The cells in the spectrometer must be made of pure silica for

ultraviolet spectra because soda glass absorbs below 365 nm, and
pyrex glass below 320 nm.
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 UV-Visible Spectrometer (4)
• Detection of the radiation passing through the sample or reference cell
can be achieved by either a photomultiplier or a photodiode,

that converts photons of radiation into tiny electrical currents;

or a semiconducting cell (that emits electrons when radiation is incident on it)
followed by an electron multiplier similar to those used in mass
spectrometers.

• The spectrum is produced by comparing the currents generated by the

sample and the reference beams.

• Wavelength checks are made by passing the sample beam through glass
samples (containing holmium oxide) that have precise absorption peaks,

The absorption is calibrated by passing the sample beam through either a
series of filters, each with a specific and known absorption, or a series of
standard solutions.

Holmium oxide: have a series of sharp optical absorption peaks in the visible
range. Used as a convenient calibration standard for optical spectrometers.

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 UV-Visible Spectrometer (5)
• For liquids, the sample is held in an optically flat, transparent container called a cell or cuvette.

• The reference cell/cuvette contains the solvent in which the sample is dissolved, commonly
referred to as the blank.

• For each wavelength the intensity of light passing through both a reference cell (I0) and the
sample cell (I) is measured.

If I is less than Io, then the sample has absorbed some of the light.

The absorbance (A) of the sample is related to I and Io according to the following equation:

I0
A = log10
I
• The detector converts the incoming light into a current.

the higher the current, the greater the intensity.

• The chart recorder usually plots the absorbance against wavelength (nm) in the UV and visible
section of the electromagnetic spectrum. (Note: absorbance does not have any units).

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 The Beer-Lambert Law
• Absorbance is proportional to concentration of the substance in solution.

ONLY true for DILUTE solutions!

I0
A = log10 = ε C l
I
• Where:

A : absorbance

l : optical path length, i.e. dimension of the cell/cuvette (cm)

C : concentration of solution (mol L-1)

ε : a constant for each absorbing material, known as the molar absorption
coefficient , (unit: L mol-1 cm-1, but by convention the units are not quoted).

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 Calibration graph

• Absorbance vs. known

concentrations of standard
solutions.

• Linear if Beer-Lambert Law is

obeyed.

• Calibration graph then used to

determine the concentration of
unknown samples.

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Example: UV-Vis Spectrum: buta-1,3-diene
• One can readily see what
wavelengths of light are absorbed
(peaks), and what wavelengths of
light are transmitted (troughs).

The higher the value, the more of a
particular wavelength is being
absorbed.

• The absorption peak at a value of

217 nm, is in the ultra-violet region,
and so there would be no visible
sign of any light being absorbed
making buta-1,3-diene colourless.

• The wavelength that corresponds to

the highest absorption is usually
referred to as “lambda-max” (λmax).

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UV-Vis Spectrum: blue copper complex

• The complementary yellow light is absorbed.

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 Quantification by UV-Vis

1. Find λmax by running full spectrum.

2. Construct calibration curve of detector response at λmax against

known amount of compound

Use peak areas rather than peak heights.

3. Run unknown under same conditions.

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 Quantification by UV-Vis (2)
• Conditions for Beer-Lambert law to be obeyed:

Light must be monochromatic - measure at λmax

Conc. of solution must be weak

Solution must not fluoresce, must be homogenous

Solute must not be photoreactive

Solute must not react with solvent

• Construct calibration curve (usually straight line) with solutions of

known concs.

• Measure unknown under same conditions and determine conc.

from calibration curve.

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 Applications
Used extensively for the quantitative analysis of all molecules that absorb UV and visible
electromagnetic radiation.
Clinical chemistry: study of enzyme kinetics
• Enzymes cannot be studied directly, but their activity can be studied by analysing the speed of the
reactions which they catalyse.
• Reagents/labels can be attached to molecules to permit indirect detection and measurement of
enzyme activity.
• Indicator of tissue damage:
• When cells are damaged by disease, enzymes leak into the bloodstream – the amount present
indicates the severity of the tissue damage.
• Relative proportions of different enzymes can be used to diagnose disease, e.g. of the liver, pancreas
or other organs which otherwise exhibit similar symptoms.

Pharmaceutical industries: dissolution testing of tablets and products

• To guide formulation design and control product quality.
• The only test that measures the rate of in-vitro drug release as a function of time, which can reflect
either: reproducibility of the product manufacturing process, or, in limited cases, in-vivo drug release.

Biochemical and genetic fields:

• Quantification of DNA and protein/enzyme activity
• Thermal denaturation of DNA.
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 Applications (2)
Dye, ink, paint industries:
• Quality control in the development and production of reagents.

Environmental and agricultural:

• Quantification of organic materials and heavy metals in fresh water.

Research: reaction kinetics

• Rate of change in concentration of reactants or products can be
determined by measuring the increase or decrease of absorbance of
coloured solutions with time.
• Plotting absorbance vs. time: gives the orders with respect to the
reactants  rates of reactions  hence the rate equation, from which
a mechanism can be proposed.

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 Question 1:
1. Show that the Beer-Lambert’s law is additive.

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 Questions 2:
A 1.28x10-4 M solution of potassium permanganate has a
transmittance of 0.5 when measured in a 1 cm cell at 525 nm.

(a)Calculate the molar absorptivity coefficient for the permanganate at
this wavelength.

(b) If the concentration is doubled, what would be the absorbance and

percentage transmittance of the new solution?

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