3
UV-VISIBLE SPECTROSCOPY
3.2 Introduction
Both molecular and atomic species can absorb radiation in the ultraviolet-visible (UV/VIS) regions of
the electromagnetic spectrum by the absorption of energy by certain electrons in a substance. In this
case, the ultraviolet region is considered to be above 200 nm.
Different types of electrons, but not all types, can absorb UV/VIS radiation, depending on the
type of substance, as shown in Table 3.2.
TABLE 3.2 Types of electrons and their absorption behaviour
Type of substance
Ion
Covalently-bonded species
Type of electron
Absorbs?
Inner shell
No
Outer shell (valence)
Yes
Single bond
No
Double/triple bond
Yes
Unbonded
Yes
EXAMPLE 3.2
Which of the following substances absorb UV/VIS radiation?
hexane, hexene, trichloromethane, potassium chloride, iron (III) chloride, potassium nitrate
Substance
Absorb?
Reason
hexane
No
All covalent bonds are single
hexene
Yes
Electrons in C=C absorb
trichloromethane
Yes
Unbonded electrons on Cl
potassium chloride
No
Both K+ and Cl- are no valence electrons
iron (III) chloride
Yes
Fe3+ has valence electrons
potassium nitrate
Yes
One of the covalent bonds in nitrate is a double bond,
and oxygen has unbonded electrons
CLASS EXERCISE 3.2
State whether the following compounds would absorb photons from the ultraviolet-visible
region: (a) sodium chloride (b) sodium sulfate (c) copper sulfate (d) benzene
3. UV-Visible Spectroscopy
In general, the more absorbing electrons in a species, the higher the absorption wavelength will be.
Only a few absorbing electrons means that the compound will generally be colourless and absorb in
the ultraviolet region only. For a compound to be coloured, there must be some absorption in the
visible region.
CLASS EXERCISE 3.2
Which of the compounds giving the spectra below will be coloured?
3.2 Instrumentation
Colorimeters
Colorimeters, in their most basic form, employ the human eye as a detector, and the human brain as a
means of comparing the colour and intensity of samples. Solutions of equal concentration have the
same colour and intensity.
Not surprisingly, colorimeters, such as the Lovibond comparator, suffer from several
disadvantages:
 a series of comparison standards must always be available;
 the human eye responds only to radiation within the wavelength range of 400-700 nm;
 the brain is unable to match absorbances if the solutions contain more than one coloured species;
and
 the eye is not sensitive to detect differences in intensity of less than 5%.
The analysis of chlorine in swimming pools is very often done at poolside using a colorimeter, where a
disc of standard colours (for different concentrations) is placed in one side of the instrument, and the
sample (with a colouring agent for the chlorine) placed in the other. The user looks at both cells
together and adjusts the standard disc until the colours match.
Filter Photometers
Filter photometers (sometimes incorrectly called colorimeters) are the simplest instrumental method of
determining the concentration of a coloured substance. They are only capable of measuring
absorbance, not recording a spectrum. The wavelength selector in a such an instrument is a piece of
absorbing material (eg coloured plastic) chosen to match the characteristics of the analyte. Normally,
these instruments operate only in the visible region, because of the cheaper filters in that region.
If a scanned spectrum is not required and there are no close together peaks, photometers can
provide as accurate results as more expensive and complicated, and less rugged spectrophotometers.
The design is relatively simple in either single- or double-beam form. A light source (generally
a tungsten lamp) is defined in area by a simple optical system, before passing through a filter, which
limits the wavelength range to that which will be absorbed by the sample solution. After passing
through the solution, the radiation intensity is measured by a detector.
Sci Inst Analysis (Spectro/Chrom)
3.2
3. UV-Visible Spectroscopy
These filter photometer are almost always single-beam, where the instrument is zeroed with a blank
solution, and calibrated to 100% absorbance by shutting off the light path. The sample solution is then
placed in the instrument and its absorbance measured.
Tungsten
lamp
Filter
Sample
cell
Detector
Readout
FIGURE 3.1 Standard components of a filter photometer
CHOOSING THE FILTER
The filters used in these photometers are generally supplied with the instrument, each transmitting a
different range of the visible spectrum. Selection of the proper filter is vital: the filter should transmit
(let through) the wavelengths that the analyte is capable of absorbing. Otherwise, the "excess"
radiation of wavelengths that will not be absorbed, will swamp the detection system.
In Chapter 1, you learnt about the effect of absorption of visible light on the colour of a
substance. To revise, the observed colour is the “opposite” of the absorbed colours. The colours
of the visible region and their wavelength ranges are shown below.
TABLE 3.2 Colours and wavelengths
Colour
violet
Wavelength range (nm)
400-450
blue
450-490
green
490-530
yellow
530-580
orange
580-620
red
620-800
When choosing a filter for use with a particular analyte solution, a knowledge of colour absorption and
transmission is one way of determining the correct filter to use. However, a better method is to
determine accurately the absorption spectrum of the analyte, and to use the basic rule: a filter should
transmit where the sample is absorbing.
Figure 3.2 shows the range of filters commonly available, the 600 series of numbers simply
being manufacturer's code numbers.
FIGURE 3.2 Transmission spectra for commonly available filters
Sci Inst Analysis (Spectro/Chrom)
3.3
3. UV-Visible Spectroscopy
EXAMPLE 3.2
(a) A red liquid is absorbing blue and green radiation, and transmitting red, orange and
yellow. What colour should the filter be? What numbered filter (from Figure 3.2) is
appropriate?
The filter must transmit where the sample is absorbing, ie, blue-green. The colour of a filter
is the colour that is transmits: ie blue-green. This filter is chosen to absorb the unwanted
red/orange/yellow light, and only let through the colours that the sample is absorbing.
The best filter from Figure 3.2 would be 603.
(b)
The absorption spectrum of a compound is shown below. What coloured filter would
be suitable for its analysis? What numbered filter (from Figure 3.2) is appropriate?
The substance is absorbing most strongly from 550-620nm. This covers the yellow and
orange regions of the spectrum.
Therefore, the filter would be yellow-orange.
The best filter from Figure 3.2 would be 606.
CLASS EXERCISE 3.3
(a) What is the best filter for the coloured species from Exercise 3.2?
(b)
What colour regions would blue-green copper sulfate be absorbing?
What filter (colour and code number) would be suitable for copper sulfate?
Sci Inst Analysis (Spectro/Chrom)
3.4
3. UV-Visible Spectroscopy
While filter photometers are generally small and inexpensive, the technique has also found application
in an instrument of quite the opposite characteristics. Known as an auto analyser, large numbers of
samples are loaded into a carriage which is then sampled in a set order by a "robot" arm. A single
sample is split into a number of sub-samples and passed through different channels which generate
colour (if necessary) through mixing with appropriate reagents, and then carry out a photometric
analysis using a set filter for the particular analyte. The filter system is used because it is a compact
and cheap method of wavelength selection.
The Technicon AutoAnalyser, the most widespread of this form of instrument, is an example of
a multi-channel device, where many measurements at different wavelengths are taken simultaneously.
As many as twelve channels, each measuring a different analyte, can be operated at the same time.
Auto analysers find their greatest application in pathological laboratories, where inorganic and
biochemical analytes in blood and other samples can be routinely determined. However, chemical
laboratories also use the instruments for water, soil and air analysis.
Scanning Spectrophotomers
Spectrophotometers are capable of two features not possible with filter photometers:

recording of the absorption spectrum from 200 nm to 800 nm

recording of absorbance/transmittance at one wavelength,
whereas filter photometers can only produce the light intensity across the range of wavelengths
allowed through by the chosen filter.
To allow the selection of one wavelength in spectrophotometers, the filter is replaced by a
monochromator. The general construction of a monochromator is shown in Figure 3.3.
dispersing
element
monochromatic
radiation
entrance slit
exit slit
polychromatic
radiation
FIGURE 3.3 A typical prism-based monochromator (focussing elements omitted)
The components are:

an entrance slit to take the polychromatic light (a mixture of many wavelengths, eg white light)
from the source,

a focussing lens or mirror (known as a collimator) to produce a parallel beam of radiation,

a grating or prism to disperse (split) the light into its component wavelengths,

a focussing lens which projects a series of rectangular images (corresponding to the individual
wavelengths) onto a plane surface,

a very narrow exit slit, which only allows one wavelength (monochromatic radiation) to pass
through (typically less than 1.0 nm, and often adjustable)

a motor which moves the dispersing element through different angles to allow different
wavelengths through the exit slit
Imagine someone shining a rainbow onto a paling fence from which one paling was missing. if you
were standing on the other side of the fence, the only colour you would see was the one getting
through the gap. That is how a monochromator works.
Prisms are the simplest form of dispersing elements in a monochromator, but gratings - a series
of grooves on the surface of a shiny piece of glass or metal - which causes interference in the incident
radiation, and results in different wavelengths being reflected at different angles.
Sci Inst Analysis (Spectro/Chrom)
3.5
3. UV-Visible Spectroscopy
To produce a spectrum, the instrument is set to a start wavelength, and the monochromator
motor is adjusted so that the prism/grating is angled to allow that wavelength to pass through the exit
slit. A final wavelength is also set, and when the scan starts, the motor slows steps through the
different angles until the final wavelength is reached. A reading is taken by the detector at each
wavelength, and the spectrum produced.
The radiation source needed for the ultraviolet region and the visible region differs. The
principal requirement of any radiation source in a conventional scanning spectrophotometer is that is
provides radiation across the region of interest at approximately uniform intensity. No simple source
can provide this across both the ultraviolet and visible regions. A white light source, most commonly
a tungsten lamp, is sufficient for the visible region, but the ultraviolet region requires a deuterium
gas discharge lamp.
Detectors used in conventional scanning spectrophotometers require a rapid response to the
incoming photons of light. Generally, the detector is not required to discriminate between photons of
different wavelength because the monochromator will have done this. Detectors used are commonly
semi-conductors, which produce an electrical current on absorption of light. The most common one is
called a photomultiplier tube.
Spectrophotometers come in single- and double-beam configurations, and are generally
computer-interfaced. The computer controls the scan and data collection, and in the case of a singlebeam instrument, stores the blank (or baseline) spectrum. This makes the recording of the spectrum
more automated, and hopefully simpler, if the computer program is well-written.
CLASS EXERCISE 3.4
Draw block diagrams, similar to Figure 3.1, for single- and double-beam spectrophotometers.
3.4 Sample preparation
While there are special attachments available which allow the recording of the ultraviolet-visible
absorption spectrum of a solid sample, most spectra are recorded from solutions. This is a limitation
of the technique, since there are many instances where species may not be stable (or at structurally
identical) when dissolved.
Cells
Three cell materials are commonly used: quartz, glass and plastic. Table 3.3 summarises the basic
properties of each.
Sci Inst Analysis (Spectro/Chrom)
3.6
3. UV-Visible Spectroscopy
TABLE 3.3 Properties of UV-Vis cells
Cell
material
Suitable
for UV
Suitable
for visible
Other issues
Quartz
Y
Y
do not use with conc. alkali, use
only when necessary due to cost
Glass
N
Y
do not use with conc. alkali; use
when plastic is not suitable
Plastic
N
Y
do not use with organics
Cells are commonly 1 centimetre pathlength, though shorter and longer cells are available for special
circumstances.
CLASS EXERCISE 3.5
(a) Give reasons for using a longer pathlength cell.
(b)
What cell would be the most suitable for the following situations?
(i)
recording of the spectrum in the UV region for a watersoluble analyte
recording of a spectrum in the visible region for a watersoluble analyte
recording of the spectrum in the visible region for a waterinsoluble analyte
measurement of the absorbance of a coloured analyte
which is water-soluble
measurement of the absorbance of a coloured analyte
which is water-insoluble
(ii)
(iii)
(iv)
(v)
Solvents
A solvent must not absorb strongly (less than 0.2 absorbance units) within 20 nm of the analytical
wavelength, otherwise the subtraction of reference from analyte absorption would lead to considerable
errors. Obviously, any colourless liquid is suitable in the visible region, but in the ultraviolet
region, there are considerable limitations on the range of wavelengths useable for different solvents.
Table 3.4 lists the minimum wavelength useable for a range of common solvents. This is often known
as the solvent window, the region of the spectrum through which radiation passes through
unabsorbed, i.e. where the solvent is transparent.
TABLE 3.4 Minimum wavelength useable in ultraviolet spectrophotometry for various solvents
Solvent
Minimum wavelength (nm)
Water
210
Ethanol
210
Trichloromethane
250
Cyclohexane
210
Propanone
330
Toluene
285
Sci Inst Analysis (Spectro/Chrom)
3.7
3. UV-Visible Spectroscopy
Colour Forming Reagents
Not all species absorb radiation in the visible region, of course. Many metal ions and most anions are
essentially colourless, though they may absorb ultraviolet radiation. Therefore, they cannot be
analysed by visible spectrophotometry. In the UV region, there is a lot of overlap between the spectra
of various species, meaning that where the species are likely to be found in the one sample, analysis
cannot be accurately carried out.
For these reasons, methods have been developed for many colourless analytes where they are
mixed with a colour forming reagent (CFR), a compound which reacts with the analyte to form an
intensely coloured solution (generally in the form of a complex). The CFR is chosen so that either it
does not react with other species, or it forms a different coloured complex with them (the former is far
preferable).
Examples include:

the formation of a blood-red colour between iron (III) and thiocyanate ion (SCN-),

the conversion of any manganese species to purple permanganate,

the "Molybdenum Blue" method for phosphate ion, where the analyte is treated with molybdate
ion and a reducing agent to yield a blue complex
In general, the absorbing species formed by the reaction of analyte and colour forming reagent has a
very large absorption coefficient, meaning that quite low concentrations (1-10 mg/L) can be analysed.
3.5 Analysis using UV/VIS absorption
Quantitative analysis
Absorption measurements in the UV/VIS region obeys Beer’s law, within the limitations described in
the previous chapter. Quantitative analysis is the main use of these instruments.
Qualitative analysis
Ultraviolet-visible absorption spectra are not generally regarded as particularly useful tools for
identification of analyte species. This is because they are usually very simple, the absorption bands
usually very broad, and do not vary greatly in wavelength between species of similar structure. Figure
3.4 shows the considerable similarity between the ultraviolet absorption spectra of the three isomeric
dimethylbenzenes.
FIGURE 3.4 Ultraviolet absorption spectra of (a) 1,2-, (b) 1,3- and (c) 1,4-dimethylbenzene
For organic species, the only useful information that can be obtained from an absorption
spectrum is simply whether the analyte possesses a chromophore, either unbonded electron pairs, or a
series of double and/or triple bonds, where the multiple bonds are separated by only one single carboncarbon bond. This is known as conjugation, and leads to absorption in the ultraviolet region: the
more bonds in conjugation, the higher the wavelength of the absorption maximum.
Sci Inst Analysis (Spectro/Chrom)
3.8
3. UV-Visible Spectroscopy
An example of a conjugated compound is 1,3-pentadiene, CH2=CH-CH=CH-CH3, whereas its isomer,
1,4-pentadiene, CH2=CH-CH2-CH=CH2, is not conjugated because there are two single C-C bonds
between the C=C. The former would exhibit an absorption maximum 30-40 nm higher than the latter.
Extra double bonds in conjugation would push the absorption peak further towards the visible region.
What You Need To Be Able To Do
 state what features of a molecule absorb photons of ultraviolet-visible radiation
 identify whether a substance would absorb UV/VIS radiation
 explain the difference in functions between colorimeters, filter photometers and scanning
spectrophotometers
 draw a diagram showing the components of photometers and scanning spectrophotometers
 explain how a monochromator works
 select the appropriate filter for an analysis by filter photometer
 describe the cells and solvents used in UV/VIS spectrophotometry
 outline the use of colour forming reagents
Terms And Definitions
Match the term with the definition.
1.
colorimeter
2.
filter photometer
3.
spectrophotometer
4.
baseline
5.
monochromator
6.
solvent window
7.
colour forming reagent
A.
substance added to analyte to produce colour
B.
spectrum of blank to be subtracted from sample spectrum
C.
device for measuring colour intensity using human eye as detector
D.
wavelength range suitable for use
E.
instrumental component capable of selecting a single wavelength
F.
instrument capable of recording spectrum
G.
instrument not capable of recording spectrum, but using electronic detection
Review Questions
1.
What class of compounds do NOT give an ultraviolet-visible absorption spectrum?. Explain
your answer.
2.
What circumstances would colorimetry be used in?
3.
Explain why a red filter would be used in the analysis of a blue-green solution.
4.
What filter (colour and code) from the range in Figure 3.2 would be suitable for the analysis of
purple permanganate solutions by filter photometry? Remember that purple is a mixture of
violet, blue and red light.
Sci Inst Analysis (Spectro/Chrom)
3.9
3. UV-Visible Spectroscopy
5.
Examine the absorption spectra below, and decide the appropriate filters from Figure 3.2
(colours and codes) to be used.
(a)
(b)
6.
A technician carrying a spectrophotometric analysis of paracetamol tablets incorrectly uses
methylbenzene (toluene) as a solvent. What problem would this cause, given that paracetamol
has an absorption maximum of 260 nm?
7.
What solvent would be suitable for the ultraviolet spectrum of a (i) non-polar and (ii) polar
organic compound?
8.
Why would propanone be an unsuitable solvent in an analysis of a spectrophotometric analysis
of a compound that had a maximum absorption at 250 nm?
9.
Why is ultraviolet-visible analysis excellent for quantitative analysis?
10.
What information regarding the structure of an organic compound can be gained from its
ultraviolet-visible absorption spectrum?
11.
Explain why compound I has an absorption maximum at 296 nm, while compound II, shows an
maximum at 228 nm.
O
Compound I
Sci Inst Analysis (Spectro/Chrom)
O
Compound II
3.10