Ultraviolet–visible

spectroscopy

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Beckman DU640 UV/Vis spectrophotometer

Ultraviolet–visible spectroscopy or
ultraviolet–visible spectrophotometry
(UV–Vis or UV/Vis) refers to absorption
spectroscopy or reflectance
spectroscopy in part of the ultraviolet
and the full, adjacent visible spectral
regions. This means it uses light in the
visible and adjacent ranges. The
absorption or reflectance in the visible
range directly affects the perceived color
of the chemicals involved. In this region
of the electromagnetic spectrum, atoms
and molecules undergo electronic
transitions. Absorption spectroscopy is
complementary to fluorescence
spectroscopy, in that fluorescence deals
with transitions from the excited state to
the ground state, while absorption
measures transitions from the ground
state to the excited state.[1]

Principle of ultraviolet–
visible absorption
Molecules containing bonding and non-
bonding electrons (n-electrons) can
absorb energy in the form of ultraviolet or
visible light to excite these electrons to
higher anti-bonding molecular orbitals.[2]
The more easily excited the electrons
(i.e. lower energy gap between the HOMO
and the LUMO), the longer the wavelength
of light it can absorb. There are four
possible types of transitions (π–π*, n–π*,
σ–σ*, and n–σ*), and they can be ordered
as follows: σ–σ* > n–σ* > π–π* > n–π*.

Applications

An example of a UV/Vis readout

UV/Vis spectroscopy is routinely used in

analytical chemistry for the quantitative
determination of different analytes, such
as transition metal ions, highly
conjugated organic compounds, and
biological macromolecules.
Spectroscopic analysis is commonly
carried out in solutions but solids and
gases may also be studied.

Solutions of transition metal ions can

be colored (i.e., absorb visible light)
because d electrons within the metal
atoms can be excited from one
electronic state to another. The colour
of metal ion solutions is strongly
affected by the presence of other
species, such as certain anions or
ligands. For instance, the colour of a
dilute solution of copper sulfate is a
very light blue; adding ammonia
intensifies the colour and changes the
wavelength of maximum absorption
(λmax).
Organic compounds, especially those
with a high degree of conjugation, also
absorb light in the UV or visible regions
of the electromagnetic spectrum. The
solvents for these determinations are
often water for water-soluble
compounds, or ethanol for organic-
soluble compounds. (Organic solvents
may have significant UV absorption;
not all solvents are suitable for use in
UV spectroscopy. Ethanol absorbs very
weakly at most wavelengths.) Solvent
polarity and pH can affect the
absorption spectrum of an organic
compound. Tyrosine, for example,
 increases in absorption maxima and
molar extinction coefficient when pH
increases from 6 to 13 or when solvent
polarity decreases.
While charge transfer complexes also
give rise to colours, the colours are
often too intense to be used for
quantitative measurement.

The Beer–Lambert law states that the

absorbance of a solution is directly
proportional to the concentration of the
absorbing species in the solution and the
path length.[3] Thus, for a fixed path
length, UV/Vis spectroscopy can be used
to determine the concentration of the
absorber in a solution. It is necessary to
know how quickly the absorbance
changes with concentration. This can be
taken from references (tables of molar
extinction coefficients), or more
accurately, determined from a calibration
curve.

A UV/Vis spectrophotometer may be

used as a detector for HPLC. The
presence of an analyte gives a response
assumed to be proportional to the
concentration. For accurate results, the
instrument's response to the analyte in
the unknown should be compared with
the response to a standard; this is very
similar to the use of calibration curves.
The response (e.g., peak height) for a
particular concentration is known as the
response factor.

The wavelengths of absorption peaks

can be correlated with the types of bonds
in a given molecule and are valuable in
determining the functional groups within
a molecule. The Woodward–Fieser rules,
for instance, are a set of empirical
observations used to predict λmax, the
wavelength of the most intense UV/Vis
absorption, for conjugated organic
compounds such as dienes and ketones.
The spectrum alone is not, however, a
specific test for any given sample. The
nature of the solvent, the pH of the
solution, temperature, high electrolyte
concentrations, and the presence of
interfering substances can influence the
absorption spectrum. Experimental
variations such as the slit width (effective
bandwidth) of the spectrophotometer will
also alter the spectrum. To apply UV/Vis
spectroscopy to analysis, these variables
must be controlled or accounted for in
order to identify the substances
present.[4]

The method is most often used in a

quantitative way to determine
concentrations of an absorbing species
in solution, using the Beer–Lambert law:

,
where A is the measured absorbance (in
Absorbance Units (AU)), is the
intensity of the incident light at a given
wavelength, is the transmitted intensity,
L the path length through the sample, and
c the concentration of the absorbing
species. For each species and
wavelength, ε is a constant known as the
molar absorptivity or extinction
coefficient. This constant is a
fundamental molecular property in a
given solvent, at a particular temperature
and pressure, and has units of
.

The absorbance and extinction ε are

sometimes defined in terms of the
natural logarithm instead of the base-10
logarithm.

The Beer–Lambert Law is useful for

characterizing many compounds but
does not hold as a universal relationship
for the concentration and absorption of
all substances. A 2nd order polynomial
relationship between absorption and
concentration is sometimes encountered
for very large, complex molecules such
as organic dyes (Xylenol Orange or
Neutral Red, for example).

UV–Vis spectroscopy is also used in the

semiconductor industry to measure the
thickness and optical properties of thin
films on a wafer. UV–Vis spectrometers
are used to measure the reflectance of
light, and can be analyzed via the
Forouhi–Bloomer dispersion equations
to determine the Index of Refraction (n)
and the Extinction Coefficient (k) of a
given film across the measured spectral
range.

Practical considerations

The Beer–Lambert law has implicit

assumptions that must be met
experimentally for it to apply; otherwise
there is a possibility of deviations from
the law.[5] For instance, the chemical
makeup and physical environment of the
sample can alter its extinction
coefficient. The chemical and physical
conditions of a test sample therefore
must match reference measurements for
conclusions to be valid. Worldwide,
pharmacopoeias such as the American
(USP) and European (Ph. Eur.)
pharmacopeias demand that
spectrophotometers perform according
to strict regulatory requirements
encompassing factors such as stray
light[6] and wavelength accuracy[7].

Spectral bandwidth

It is important to have a monochromatic

source of radiation for the light incident
on the sample cell.[5] Monochromaticity
is measured as the width of the "triangle"
formed by the intensity spike, at one half
of the peak intensity. A given
spectrometer has a spectral bandwidth
that characterizes how monochromatic
the incident light is. If this bandwidth is
comparable to (or more than) the width
of the absorption line, then the measured
extinction coefficient will be mistaken. In
reference measurements, the instrument
bandwidth (bandwidth of the incident
light) is kept below the width of the
spectral lines. When a test material is
being measured, the bandwidth of the
incident light should also be sufficiently
narrow. Reducing the spectral bandwidth
reduces the energy passed to the
detector and will, therefore, require a
longer measurement time to achieve the
same signal to noise ratio.

Wavelength error

In liquids, the extinction coefficient

usually changes slowly with wavelength.
A peak of the absorbance curve (a
wavelength where the absorbance
reaches a maximum) is where the rate of
change in absorbance with wavelength is
smallest.[5] Measurements are usually
made at a peak to minimize errors
produced by errors in wavelength in the
instrument, that is errors due to having a
different extinction coefficient than
assumed.
Stray light

Another important factor is the purity of

the light used. The most important factor
affecting this is the stray light level of the
monochromator.[5]

The detector used is broadband; it

responds to all the light that reaches it. If
a significant amount of the light passed
through the sample contains
wavelengths that have much lower
extinction coefficients than the nominal
one, the instrument will report an
incorrectly low absorbance. Any
instrument will reach a point where an
increase in sample concentration will not
result in an increase in the reported
absorbance, because the detector is
simply responding to the stray light. In
practice the concentration of the sample
or the optical path length must be
adjusted to place the unknown
absorbance within a range that is valid
for the instrument. Sometimes an
empirical calibration function is
developed, using known concentrations
of the sample, to allow measurements
into the region where the instrument is
becoming non-linear.

As a rough guide, an instrument with a

single monochromator would typically
have a stray light level corresponding to
about 3 Absorbance Units (AU), which
would make measurements above about
2 AU problematic. A more complex
instrument with a double monochromator
would have a stray light level
corresponding to about 6 AU, which
would therefore allow measuring a much
wider absorbance range.

Deviations from the Beer–Lambert law

At sufficiently high concentrations, the

absorption bands will saturate and show
absorption flattening. The absorption
peak appears to flatten because close to
100% of the light is already being
absorbed. The concentration at which
this occurs depends on the particular
compound being measured. One test that
can be used to test for this effect is to
vary the path length of the measurement.
In the Beer–Lambert law, varying
concentration and path length has an
equivalent effect—diluting a solution by a
factor of 10 has the same effect as
shortening the path length by a factor of
10. If cells of different path lengths are
available, testing if this relationship holds
true is one way to judge if absorption
flattening is occurring.

Solutions that are not homogeneous can

show deviations from the Beer–Lambert
law because of the phenomenon of
absorption flattening. This can happen,
for instance, where the absorbing
substance is located within suspended
particles (see "Beer's law revisited",
Berberan-Santos, J. Chem. Educ. 67
(1990) 757, and "Absorption flattening in
the optical spectra of liposome-
entrapped substances", Wittung, Kajanus,
Kubista, Malmström, FEBS Lett 352
(1994) 37). The deviations will be most
noticeable under conditions of low
concentration and high absorbance. The
last reference describes a way to correct
for this deviation.

Some solutions, like copper(II)chloride in

water, change visually at a certain
concentration because of changed
conditions around the coloured ion (the
divalent copper ion). For
copper(II)chloride it means a shift from
blue to green,[8] which would mean that
monochromatic measurements would
deviate from the Beer–Lambert law.

Measurement uncertainty sources

The above factors contribute to the

measurement uncertainty of the results
obtained with UV/Vis spectrophotometry.
If UV/Vis spectrophotometry is used in
quantitative chemical analysis then the
results are additionally affected by
uncertainty sources arising from the
nature of the compounds and/or
solutions that are measured. These
include spectral interferences caused by
absorption band overlap, fading of the
color of the absorbing species (caused
by decomposition or reaction) and
possible composition mismatch between
the sample and the calibration solution.[9]

Ultraviolet–visible
spectrophotometer
The instrument used in ultraviolet–visible
spectroscopy is called a UV/Vis
spectrophotometer. It measures the
intensity of light passing through a
sample ( ), and compares it to the
intensity of light before it passes through
the sample ( ). The ratio is called
the transmittance, and is usually
expressed as a percentage (%T). The
absorbance, , is based on the
transmittance:

The UV–visible spectrophotometer can

also be configured to measure
reflectance. In this case, the
spectrophotometer measures the
intensity of light reflected from a sample
( ), and compares it to the intensity of
light reflected from a reference material (
) (such as a white tile). The ratio
is called the reflectance, and is usually
expressed as a percentage (%R).
The basic parts of a spectrophotometer
are a light source, a holder for the
sample, a diffraction grating in a
monochromator or a prism to separate
the different wavelengths of light, and a
detector. The radiation source is often a
Tungsten filament (300–2500 nm), a
deuterium arc lamp, which is continuous
over the ultraviolet region (190–400 nm),
Xenon arc lamp, which is continuous
from 160 to 2,000 nm; or more recently,
light emitting diodes (LED)[1] for the
visible wavelengths. The detector is
typically a photomultiplier tube, a
photodiode, a photodiode array or a
charge-coupled device (CCD). Single
photodiode detectors and
photomultiplier tubes are used with
scanning monochromators, which filter
the light so that only light of a single
wavelength reaches the detector at one
time. The scanning monochromator
moves the diffraction grating to "step-
through" each wavelength so that its
intensity may be measured as a function
of wavelength. Fixed monochromators
are used with CCDs and photodiode
arrays. As both of these devices consist
of many detectors grouped into one or
two dimensional arrays, they are able to
collect light of different wavelengths on
different pixels or groups of pixels
simultaneously.
Simplified schematic of a double beam UV–visible
spectrophotometer

A spectrophotometer can be either single

beam or double beam. In a single beam
instrument (such as the Spectronic 20),
all of the light passes through the sample
cell. must be measured by removing
the sample. This was the earliest design
and is still in common use in both
teaching and industrial labs.

In a double-beam instrument, the light is

split into two beams before it reaches the
sample. One beam is used as the
reference; the other beam passes
through the sample. The reference beam
intensity is taken as 100% Transmission
(or 0 Absorbance), and the measurement
displayed is the ratio of the two beam
intensities. Some double-beam
instruments have two detectors
(photodiodes), and the sample and
reference beam are measured at the
same time. In other instruments, the two
beams pass through a beam chopper,
which blocks one beam at a time. The
detector alternates between measuring
the sample beam and the reference
beam in synchronism with the chopper.
There may also be one or more dark
intervals in the chopper cycle. In this
case, the measured beam intensities may
be corrected by subtracting the intensity
measured in the dark interval before the
ratio is taken.

In a single-beam instrument, the cuvette

containing only a solvent has to be
measured first. Mettler Toledo developed
a single beam array spectrophotometer
that allows fast and accurate
measurements over the UV/VIS range.
The light source consists of a Xenon
flash lamp for the ultraviolet (UV) as well
as for the visible (VIS) and near-infrared
wavelength regions covering a spectral
range from 190 up to 1100 nm. The lamp
flashes are focused on a glass fiber
which drives the beam of light onto a
cuvette containing the sample solution.
The beam passes through the sample
and specific wavelengths are absorbed
by the sample components. The
remaining light is collected after the
cuvette by a glass fiber and driven into a
spectrograph. The spectrograph consists
of a diffraction grating that separates the
light into the different wavelengths, and a
CCD sensor to record the data,
respectively. The whole spectrum is thus
simultaneously measured, allowing for
fast recording[10].
Samples for UV/Vis spectrophotometry
are most often liquids, although the
absorbance of gases and even of solids
can also be measured. Samples are
typically placed in a transparent cell,
known as a cuvette. Cuvettes are
typically rectangular in shape, commonly
with an internal width of 1 cm. (This
width becomes the path length, , in the
Beer–Lambert law.) Test tubes can also
be used as cuvettes in some
instruments. The type of sample
container used must allow radiation to
pass over the spectral region of interest.
The most widely applicable cuvettes are
made of high quality fused silica or
quartz glass because these are
transparent throughout the UV, visible and
near infrared regions. Glass and plastic
cuvettes are also common, although
glass and most plastics absorb in the UV,
which limits their usefulness to visible
wavelengths.[1]

Specialized instruments have also been

made. These include attaching
spectrophotometers to telescopes to
measure the spectra of astronomical
features. UV–visible
microspectrophotometers consist of a
UV–visible microscope integrated with a
UV–visible spectrophotometer.

A complete spectrum of the absorption

at all wavelengths of interest can often
be produced directly by a more
sophisticated spectrophotometer. In
simpler instruments the absorption is
determined one wavelength at a time and
then compiled into a spectrum by the
operator. By removing the concentration
dependence, the extinction coefficient (ε)
can be determined as a function of
wavelength.

Microspectrophotometry
UV–visible spectroscopy of microscopic
samples is done by integrating an optical
microscope with UV–visible optics, white
light sources, a monochromator, and a
sensitive detector such as a charge-
coupled device (CCD) or photomultiplier
tube (PMT). As only a single optical path
is available, these are single beam
instruments. Modern instruments are
capable of measuring UV–visible spectra
in both reflectance and transmission of
micron-scale sampling areas. The
advantages of using such instruments is
that they are able to measure
microscopic samples but are also able to
measure the spectra of larger samples
with high spatial resolution. As such, they
are used in the forensic laboratory to
analyze the dyes and pigments in
individual textile fibers,[11] microscopic
paint chips [12] and the color of glass
fragments. They are also used in
materials science and biological
research and for determining the energy
content of coal and petroleum source
rock by measuring the vitrinite
reflectance. Microspectrophotometers
are used in the semiconductor and micro-
optics industries for monitoring the
thickness of thin films after they have
been deposited. In the semiconductor
industry, they are used because the
critical dimensions of circuitry is
microscopic. A typical test of a
semiconductor wafer would entail the
acquisition of spectra from many points
on a patterned or unpatterned wafer. The
thickness of the deposited films may be
calculated from the interference pattern
of the spectra. In addition, ultraviolet–
visible spectrophotometry can be used to
determine the thickness, along with the
refractive index and extinction coefficient
of thin films as described in Refractive
index and extinction coefficient of thin
film materials. A map of the film
thickness across the entire wafer can
then be generated and used for quality
control purposes.[13]

Additional applications
UV/Vis can be applied to determine the
kinetics or rate constant of a chemical
reaction. The reaction, occurring in
solution, must present color or
brightness shifts from reactants to
products in order to use UV/Vis for this
application.[2] For example, the molecule
mercury dithizonate is a yellow-orange
color in diluted solution (1*10^-5 M), and
turns blue when subjected with particular
wavelengths of visible light (and UV) via a
conformational change, but this reaction
is reversible back into the yellow "ground
state".[14]

The rate constant of a particular reaction

can be determined by measuring the
UV/Vis absorbance spectrum at specific
time intervals. Using mercury dithizonate
again as an example, one can shine light
on the sample to turn the solution blue,
then run a UV/Vis test every 10 seconds
(variable) to see the levels of absorbed
and reflected wavelengths change over
time in accordance with the solution
turning back to yellow from the excited
blue energy state. From these
measurements, the concentration of the
two species can be calculated.[15] The
mercury dithizonate reaction from one
conformation to another is first order and
would have the integral first order rate
law : ln[A](time t)=−kt+ln[A](initial).
Therefore, graphing the natural log (ln) of
the concentration [A] versus time will
graph a line with slope -k, or negative the
rate constant. Different rate orders have
different integrated rate laws depending
on the mechanism of the reaction.

An equilibrium constant can also be

calculated with UV/Vis spectroscopy.
After determining optimal wavelengths
for all species involved in equilibria, a
reaction can be run to equilibrium, and
the concentration of species determined
from spectroscopy at various known
wavelengths. The equilibrium constant
can be calculated as K(eq) = [Products] /
[Reactants].

See also
Isosbestic point important in kinetics
measurements. A wavelength where
absorption does not change as the
reaction proceeds.
Ultraviolet–visible spectroscopy of
stereoisomers
Infrared spectroscopy and Raman
spectroscopy are other common
spectroscopic techniques, usually
used to obtain information about the
structure of compounds or to identify
compounds. Both are forms of
vibrational spectroscopy.
Fourier-transform spectroscopy
Near-infrared spectroscopy
Vibrational spectroscopy
Rotational spectroscopy
Applied spectroscopy
 Slope spectroscopy
Benesi–Hildebrand method
Spectrophotometry
DU spectrophotometer – first UV–Vis
instrument
Charge modulation spectroscopy

References
1. Skoog, Douglas A.; Holler, F. James;
Crouch, Stanley R. (2007). Principles
of Instrumental Analysis (6th ed.).
Belmont, CA: Thomson Brooks/Cole.
pp. 169–173. ISBN 9780495012016.
2. Metha, Akul (13 December 2011).
"Principle" . [Link].
3. Metha, Akul (22 April 2012).
"Derivation of Beer–Lambert Law" .
[Link].
4. Misra, Prabhakar; Dubinskii, Mark,
eds. (2002). Ultraviolet Spectroscopy
and UV Lasers. New York: Marcel
Dekker. ISBN 978-0-8247-0668-5.
5. Metha, Akul (14 May 2012).
"Limitations and Deviations of Beer–
Lambert Law" .
[Link].
6. "Stray Light and Performance
Verification" .
7. "Wavelength Accuracy in UV/VIS
Spectrophotometry" .
8. Ansell, S.; Tromp, R. H.; Neilson, G.
W. (1995). "The solute and aquaion
structure in a concentrated aqueous
solution of copper(II) chloride". J.
Phys.: Condens. Matter. 7 (8): 1513–
1524.
Bibcode:1995JPCM....7.1513A .
doi:10.1088/0953-8984/7/8/002 .
9. Sooväli, L.; Rõõm, E.-I.; Kütt, A.; et al.
(2006). "Uncertainty sources in UV–
Vis spectrophotometric
measurement". Accreditation and
Quality Assurance. 11 (5): 246–255.
doi:10.1007/s00769-006-0124-x .
10. reserved, Mettler-Toledo
International Inc. all rights.
"Spectrophotometry Applications and
Fundamentals" . [Link].
Retrieved 10 July 2018.
11. Forensic Fiber Examination
Guidelines, Scientific Working Group-
Materials, 1999,
[Link]
12. Standard Guide for
Microspectrophotometry and Color
Measurement in Forensic Paint
Analysis, Scientific Working Group-
Materials, 1999,
[Link]
13. "Spectroscopic thin film thickness
measurement system for
semiconductor industries", Horie, M.;
Fujiwara, N.; Kokubo, M.; Kondo, N.,
Proceedings of Instrumentation and
Measurement Technology
Conference, Hamamatsu, Japan,
1994,(ISBN 0-7803-1880-3).
14. Sertova (June 2000).
"Photochromism of mercury(II)
dithizonate in solution". Journal of
Photochemistry and Photobiology A:
Chemistry. 134 (3): 163–168.
doi:10.1016/s1010-6030(00)00267-
7.
 15. UC Davis (2 October 2013). "The
Rate Law" . ChemWiki. Retrieved
11 November 2014.

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