Ultraviolet–visible spectroscopy:-

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.

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.
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:-
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. 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.
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 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 1/M*cm.

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. 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.

Spectral bandwidth:-
It is important to have a monochromatic source of radiation for the
light incident on the sample cell. 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.[clarification
needed] 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. 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.

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. 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. 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.

Ultraviolet–visible spectrophotometer:-

The instrument used in ultraviolet–visible spectroscopy is called a

UV/Vis spectrophotometer. It measures the intensity of light after
passing through a ssampl 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, A, 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
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.
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.

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, L, 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.

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.