Chapter 13- An Introduction to Ultraviolet/Visible Molecular Absorption

Spectrometry
A: Measurement of Transmittance and Absorbance
An introduction into any field requires that one learn the terms and symbols associated
with work in that field. Unfortunately, the terms used in spectroscopy and
spectrophotometry are somewhat confusing. The common terms, symbols, and
definitions employed in the measurement of absorption are listed in the table below. The
recommended terms and symbols are listed under the column labeled Term and Symbol.
(Principles of Instrumental Analysis)

The letters P and Po of the above table represent the power of a beam of light before and
after passage through an absorbing species respectively. Absorbance, like the table shows,
can be defined as the base-ten logarithm of the reciprocal of the transmittance:
A = log 1/T = -log I/Io = -log P/Po
It is important to note that the absorbance of a solution increases as the attenuation of the
beam becomes greater. Reflection and scattering losses are significant and to compensate
for these effects, the power of the beam transmitted by the analyte solution is ordinarily
compared with the power of the beam transmitted by an identical cell containing only the
solvent. An experimental absorbance that closely approximates the true absorbance is
then obtained with the equation
A = log Psolvent/Psolution ~ log Po/P

The 0%T adjustment is performed with the detector screened from the source by a
mechanical shutter. In this adjustment, the instrument is thus counter signaled so as to
read zero in the absence of any radiation from the source and the dark current that many
detectors exhibit in the absence of radiation is eliminated. The 100% adjustment is made
with the shutter open and the solvent in its light path. Normally the solvent is contained
in a cell that is as nearly as possible identical to the one containing the samples. The
100% adjustment may involve increasing or decreasing the radiation output of the source
electrically; alternatively, the power of the beam may be varied with an adjustable
diaphragm or by appropriate positioning of a comb or optical wedge, which attenuates the
beam to a varying degree depending upon its position with respect to the beam.
B: Beers Law
Bouguer, and later Lambert, observed that the fraction of the energy, or the intensity, of
radiation absorbed in a thin layer of material depends on the absorbing substance and on
the frequency of the incident radiation, and is proportional to the thickness of the layer.
At a given concentration of the absorbing species, summation over a series of thin layers,
or integration over a finite thickness, leads to an exponential relationship between
transmitted intensity and thickness. This is generally called Lamberts law. Beer showed
that, at a given thickness, the absorption coefficient introduced by Lamberts law was
directly proportional to the concentration of the absorbing substance in a solution.
Combination of these two results gives the relationship now commonly known as Beers
law. This law states that the amount of radiation absorbed or transmitted by a solution or
medium is an exponential function of the concentration of the absorbing substance
present and of the length of the path of the radiation through the sample. Beers law can
be derived as follows. Consider the block of absorbing matter (solid, liquid, or gas)
shown in the figure below. A parallel beam of monochromatic radiation with power Po
strikes the block perpendicular to a surface; after passing through a length b of the
material, which contains n absorbing particles, the beams power is decreased to P as a
result of absorption. Consider now a cross-section of the block having an area S and an
infinitesimal thickness dx. Within this section there are dn absorbing particles; associated
with each particle we can imagine a surface at which photon capture will occur. That is, if
a photon reaches one of these areas by chance it will be absorbed. The total projected area
of these capture surfaces within the section is designated as dS; the ratio of the capture
area to the total area, then, is dS/S. On a statistical average this ratio represents the
probability for the capture of photons within this section.

The power of the beam entering the section, Px, is proportional to the number of
photons per square centimeter per second, and dPx represents the quantity removed per
second within the section; the fraction absorbed is then - dPx/Px, and this ratio also
equals the average probability for capture. The term is given a minus sign to indicate that
P undergoes a decrease. Thus,
- dPx/Px = dS/S
Since dS is the sum of the capture areas present in section, dS must be proportional to the
number of particles or dS = dn where dn is the number of particles and is the
proportionality constant. Substitution into the previous equation and integrating from 0 to
n yields
- In P/Po = n/S
Upon converting to base ten logarithms and inverting to change the sign, we obtain
log Po/P = n/2.303S
Since S (i.e. area under consideration) can be written as V/b where V is the volume of the
block then the last equation can be written as
log Po/P = nb/2.303V
Noting that n/V has the units of concentration i.e. number of particles per cubic
centimeter, conversion to moles per liter yields the following equation
log Po/P = NA*bc/2.303 1000
where NA is Avogradros number. Finally, collecting the constants into a single term
gives
log Po/P = bc = A
The derivation of this law assumes (a) that the incident radiation is monochromatic, (b)
the absorption occurs in a volume of uniform cross-section, and (c) the absorbing
substances behave independently of each other in the absorbing process. Thus, when
Beers law applies to a multi component system in which there is no interaction among
the various species, the total absorbance may be expressed as
Atotal = 1bc1 + 2bc2 + .........+ nbcn
Applications of Beers Law to Mixtures
Beers law also applies to a medium containing more than one kind of absorbing
substance. Provided that there is no interaction among the various species, the total
absorbance for a multicomponent system is given by
Atot = A1 + A2 + + An
Where the subscripts refer to absorbing components 1, 2, , n.
Limitations to Beers Law
Few exceptions are found to the generalization that absorbance is linearly related to path
length. On the other hand, deviations from the direct proportionally between the
measured absorbance and concentration when b is constant are frequently encountered.
Instrumental deviations
Chemical deviations

Real Limitations to Beers Law

Beers law is successful in describing the absorption behavior of dilute solutions only; in
this sense it is a limiting law. At high concentrations (> 0.01M), the average distance
between the species responsible for absorption is diminished to the point where each
affects the charge distribution of its neighbors. This interaction, in turn, can alter the
species ability to absorb at a given wavelength of radiation thus leading to a deviation
from Beers law. Deviations also arise because is dependent upon the refractive index
of the solution. Thus, if concentration changes cause significant alterations in the
refractive index of a solution, departures from Beers law are observed. It is not
which is constant and independent of concentration, but the expression
= true* /( + 2)
where is the refractive index of the solution. At concentrations of 0.01 or less, the
refractive index is essentially constant, but at high concentrations the refractive index
may vary considerably and so will . This does not rule out quantitative analyses at high
concentrations, since bracketing standard solutions and a calibration curve can provide
sufficient accuracy.
Apparent Chemical Deviations
Chemical deviations from Beers law are caused by shifts in the position of a chemical or
physical equilibrium involving the absorbing species. A common example of this
behavior is found with acid/base indicators. Deviations arising from chemical factors can
only be observed when concentrations are changed.
Apparent Instrumental Deviations with Polychromatic Radiation
Unsatisfactory performance of an instrument may be caused by fluctuations in the powersupply voltage, an unstable light source, or a non-linear response of the detector-amplifier
system. In addition the following instrumental sources of possible deviations should be
understood:
Polychromatic radiation. Strict adherence to Beers law is observed only with truly
monochromatic radiation. This sort of radiation is only approached in specialized line
emission sources. All monochromators, regardless of quality and size, have a finite
resolving power and therefore minimum instrumental bandwidth. A good picture of the
effect of polychromatic radiation can be presented as follows. When radiation consists of
two wavelengths, and 1, and assuming that Beers law applies at each of these
individually the absorbance at is given by
log ( Po/P ) = A = bc
Po/P = 10bc
Similarly, at 1,
P1o/P1 = 101bc
The radiant power of two wavelengths passing through the solvent is given by Po
+ P1o, and that passing through the solution containing absorbing species by P + P1. The
combined absorbance is
Ac = log ( Po + P1o)/P + P1
Substituting for P and P1, we obtain
Ac = log (Po + P1o)/(Po10-bc + P1o10-1bc)

In the very special case where 1 = , the above equation becomes Beers law. The
relationship between Ac and concentration is no longer linear when the molar
absorptivities differ; moreover, greater departures from linearity can be expected with
increasing differences between 1 and It is also found that deviations from Beers law
resulting from the use of a polychromatic beam are not appreciable, provided the
radiation used does not encompass a spectral region in which the absorber does not
exhibit large changes in absorption as a function of wavelength.
Instrumental Deviations in the Presence of Stray Radiation
Stray light affects absorption measurements because stray radiation often differs in
wavelength from that of the principal radiation and, in addition, may not have passed
through the sample. When measurements are made in the presence of stray radiation, the
observed absorbance is given by
A = log( Po + Ps)/(P + Ps)
where Ps is the power of nonabsorbed stray radiation. It has been deduced that positive
deviations from Beers law occurs when stray radiation is absorbed, and negative
deviation if it is not.
C: The Effects of Instrumental Noise on Spectrophotometric Analyses
Instrumental Noise as a Function of Transmittance
A spectrophotometric measurement entails three steps:
A 0% T adjustment
A 100% T adjustment
A measurement of % T with the sample in the radiation path
The noise associated with each of these steps combines to give a net uncertainty for the
final value obtained for T. The relationship between the noise encountered in the
measurement of T and the uncertainty in concentration can be derived by writing Beers
law in the form
c = -(1/b)log T = -(0.434/b)ln T
Types of Noise
Shot noise This noise is generated by current flowing across a P-N junction and
is a function of the bias current and the electron charge. The impulse of charge q
depicted as a single shot event in the time domain can be Fourier transformed into
the frequency domain as a wideband noise.
Thermal noise In any object with electrical resistance the thermal fluctuations
of the electrons in the object will generate noise.
White noise- The spectral density of thermal noise is flat with frequency.
Burst noise Occurs in semiconductor devices, especially monolithic amplifiers
and manifests as a noise crackle.
Avalanche noise Occurs in Zener diodes are reversed biased P-N junctions at
breakdown. This noise is considerably larger than shot noise, so if zeners have to
be used as part of a bias circuit then they need to be RF decoupled.

Flicker noise This noise occurs in almost all electronic devices at low
frequencies. Flicker noise is usually defined by the corner frequency FL.
Sources of Instrumental Noise
Case I: sT = k1
Case II: sT = k2(T2 + T)
Case III: sT = k3T
Effect of Slit Width on Absorbance Measurements
The ability of a spectrometer to distinguish between two frequencies differing only
slightly from each other depends upon the widths of the images produced (relative to the
separation of the two images). The width of the image produced is thus an important
measure of the quality of the performance of a spectrometer. The figure below shows the
loss of detail that accompanies the use of wider slits. It is evident that an increase in slit
width brings about a loss of spectral detail.

Another effect of slit width is the change of absorbance values that accompany a change
in the slit width. The figure below illustrates this effect. Note that the peak absorbance
values increase significantly (by as much as 70% in one instance) as the slit width
decreases.

It is evident from both these illustrations that quantitative measurement of narrow

absorption bands demand the use of narrow slits widths. Unfortunately, a decrease in slit
width is accompanied by a second-order power reduction in the radiant energy; at very
narrow settings spectral detail may be lost owing to an increase in the signal-to-noise
ratio. In general, it is good practice to narrow slits no more than is necessary for good
resolution for the spectrum at hand.
D: Instrumentation
Instrument Components
Instruments used for measuring the absorption of ultra-violet, visible and infrared
radiation are made up of one or more (1) sources, (2) wavelengths selectors (3) sample
containers, (4) radiation detectors, and (5) signal processors and readout devices.
Sources
There are several light sources available for use in the ultraviolet-visible region. For the
purpose of molecular absorption a continuous source is required whose power does not
change sharply over a considerable range of wavelengths.
Deuterium and Hydrogen Lamps
An important feature of deuterium and hydrogen discharge lamps is the shape of aperture
between the two electrodes, which constricts the discharge to a narrow path. As a
consequence, an intense ball of radiation about 1 to 1.5mm in diameter is produced.
Deuterium gives a somewhat larger and brighter ball than hydrogen, which accounts for

the widespread use of the former. They both produce a useful continuum spectrum in the
region of 160 to 375nm. At longer wavelengths, the lamps produce emission lines, which
are superimposed on the sontinuum spectrum. For many applications, these lines
represent a nuisance; they can be useful, however, for wavelength calibration of
absorption instruments.

Probably a deuterium lamp

(about 3 inches long). The
tube was sealed into a
metal tube and clamped to
a heatsink. One reason for
the sealing is probably to
reduce ozone, which can
be smelt strongly after the
tube is run for just a few
seconds.

Tungsten Filament Lamps

Tungsten-filament incandescent lamps are commonly used in the visible and nearinfrared region. These are thermal or black body sources in which the radiation is the
result of high temperature of the solid filament material. These sources provide
continuous radiation from about 320 to 3000 nm - most of it, unfortunately in the near
infrared. At the usual operating temperature of about 3000 K, only about 155 of the total
radiant energy falls in the visible region. The lifetime of a tungsten filament lamp can be
greatly increased by the presence of a low pressure of iodine or bromine vapor within the
lamp. Most work in the ultraviolet region is done with hydrogen or deuterium electricaldischarge lamps typically operated under low-pressure DC conditions (about 40 V with 5
mm gas pressure). These lamps provide a continuous source of radiation form 375 nm
down to about 160 nm hence quartz windows must be used with these types of lamps
since glass absorbs strongly at wavelengths less than about 350 nm.

Tungsten Filament Lamps

This lamp produces intense radiation by the passage of current through an atmosphere of
xenon. The spectrum is continuous over the range between about 200 to 1000nm, with
the peak intensity occurring at about 500nm.

Sample Containers
In common with other optical elements of an absorption instrument, the cells, or cuvettes
that hold the sample must be constructed of a material that passes radiation in the spectral
region of interest. The best cells have windows that are perfectly normal to the direction
of the beam in order to minimize reflection losses.
Types of Instruments
Four general types of spectroscopic instruments will be discussed:
Single-beam
Double-beam in space
Double-beam in time
Multichannel
Single-Beam Instruments
The figure below contains diagrams showing instrument designs for photometers and
spectrophotometers.

The simplest type is the single-beam instrument shown as (a) in the figure below. There
is only one light beam or optical path from the source to the detector. The wavelength
selector is either a filter or monochromator and the determination of transmittance
involves three successive steps that are separated in time; (1) the 0%T setting with a
shutter in place; (2) the 100% T adjustment with the solvent in the light path and (3) the
measurement of the %T with the sample in place. In single beam instruments there is
only one light beam or optical path from the source to the detector.

Double-Beam Instruments
Alternatively, in double beam instruments (i.e. diagram (b)), the reference and the sample
may be compared several times a second. The light from the source after passing through
the monochromator, is split into two separate beams - one for the sample and the other for
reference.

Multichannel Instruments
The most recent type of spectrophotometer, which appeared on the market in the early
1980s, is a single beam instrument based upon the diode array transducer described in
Chapter 7. The entire cycle of this instrument takes a few milliseconds. The
monochromator slit width of a diode array instrument is usually made identical to the
width of one of the silicon diodes.
Some Typical Instruments
Under this heading a few of the typical photometers and spectrometers (instruments
incorporating a monochromator ) are described.
Photometers
Photometers provide relatively inexpensive tools for performing absorption analyses.
Filter photometers are often more convenient, more rugged, and easier to maintain and
use than are the more sophisticated spectrometers. The figure below presents the
schematics for two photometers. The upper figure illustrates the single-beam, direct
reading instrument consisting of a tungsten filament lamp, a lens to provide a parallel
beam of light, a filter, and a photovoltaic cell. Diagram (a) is that of a single-beam
photometer while diagram (b) is a double-beam photometer.

For the single-beam photometer, the optical path is simply from the light source, through
the filter and the sample holder, and to the detector. Light from the tungsten filament
lamp in the reflector is defined in area by fixed apertures in the sample holder and
restricted to a desired band of wavelengths by an absorption or interference filter. After
passing through the sample cuvette, the light strikes the surface of a photovoltaic cell, the
output of which is measured by the deflection of a rugged light-spot galvanometer. Yet
another type of photometer is the one illustrated below. It is classified as a probe-type
photometer and it employs an optical fiber to transmit light from a source to a layer of
solution lying between the glass seal at the end of the fiber and a mirror. The reflected
radiation from the latter passes to photo diode detector via a second glass fiber. The
photometer uses an amplifier with an electric chopper that is synchronized with the light
source; as a result, the photometer does not respond to extraneous radiation. Absorbance
is measured by dipping the probe into the solvent and then into the solution to be
measured.
Visible Photometers

Probe-Type Photometers

Filter Selection
Ultraviolet Absorption Photometers
Spectrophotometers
Several spectrometers designed to operate in the wavelength range of about 380 to 800
nm are available from commercial sources. These instruments are frequently simple,
single-beam grating instruments that are relatively inexpensive. The figure below shows
a simple and inexpensive spectrometer, the Spectronic 20. The original version of this
instrument first appeared in the market in the mid-1950s, and the modified version shown
in the figure is still being manufactured and widely sold.

1) On/Off switch and zero transmission adjustment knob

2) Wavelength selector/Readout
3) Sample chamber
4) Blank adjustment knob
5) Absorbance/Transmittance scale
Instruments for the Visible Region

Single-Beam Instruments for the Ultraviolet/Visible Region

Fig. 1
Schematic diagram of a single-beam spectrophotometer

Single-Beam Computerized Spectrophotometers

Inside of a single-beam spectrophotometer connected to a computer.

Double-Beam Instrumetents
Numerous double-beam spectrophotometers for the ultraviolet/visible region of the
spectrum are now available. Generally, these instruments are more expensive than their
single beam counterparts. A current studies involving UV-Vis spectrometry includes
Variable Path Length Transmittance Cell for Ultraviolet, Visible, and Infrared
Spectroscopy and Spectroelectrochemistry by Paul A. Flowers* and Sherry-Ann
Callender, which was found in Analytical Chemistry, 68 (1), 199 -202, 1996. The abstract
discusses the design and characteristics of a transmittance cell for ultraviolet, visible, and
infrared spectroscopy and spectroelectrochemistry. Through modification of a previously
reported design, this cell employs threaded glass connectors as insertion ports for either
quartz- or silicon-windowed tubes, thus permitting essentially continuous variation of the
optical path length from ~0.050 to 200 mm. Though the initial fabrication requires
skillful glassblowing, once constructed, the cell's simple design allows for rapid and
reproducible disassembly/reassembly between experiments. The utility of the cell for a
diversity of fluid samples is demonstrated through applications to water, aqueous
ferricyanide, ferrocene in methylene chloride, and acetone vapor. Ions were quantitated
with estimation errors below 10 mM using this procedure. In addition, the species'
spectral profiles were recovered, and optimum estimates of the system's equilibrium
constants were determined.
Double-Dispersing Instruments
Diode Array Instruments

Diode array technology is unique in the sense that it doesn't use any moving parts in the
optics. This greatly improves the stability of the instrument as there are no parts that can
be worn out or misaligned. The result is an instrument which requires much less
maintenance than when moving parts are used, as for example is the case with scanning
monochromators. The simple picture above shows the optical principle of the DA 7200.
1. A lamp illuminates the sample with white light. Some of the light
is absorbed (depending on the composition of the sample) and
the rest is reflected.
2. The light which is reflected hits a stationary grating, which
separates the light by wavelength. Instead of white light, we now
have a "rainbow".
3. Each wavelength is measured by a dedicated detector.

References
http://www.anachem.umu.se/jumpstation.htm
http://userwww.service.emory.edu/~kmurray/mslist.html
http://www.anachem.umu.se/jumpstation.htm
http://userwww.service.emory.edu/~kmurray/mslist.html
http://www.anachem.umu.se/jumpstation.htm
http://www1.shimadzu.com/products/lab/spectro/uv3150.html
http://www.perten.com/product_range/diode_array/da_technology.html
http://repairfaq.ece.drexel.edu/sam/CORD/leot/course10_mod03/mod10-03.html
http://las.perkinelmer.com/catalog/Product.aspx?ProductID=L950
http://www.olisweb.com/products/upgrades/ir983.php

http://imagers.gsfc.nasa.gov/ems/visible.html
http://biology.easternct.edu/courses/spectwenty.htm
http://www.cairnweb.com/systems/prod_lamp.html
http://www.odyseus.nildram.co.uk/RFIC_Theory_Files/Noise_Tutorial.pdf