Principle of UV-VIS Spectroscopy

The theory of UV-visible spectroscopy exploits the wave-like nature of

electromagnetic radiation and its interaction with matter and is widely used in
analytical spectroscopic instruments to identify, characterize and quantify an
extraordinary wide range of molecular compounds.

When a material is irradiated with an electromagnetic wave, phenomena such

as transmission, absorption, reflection, and scattering can occur and the
observed spectrum shows the interaction of wavelengths with objects of a
discrete dimensions, such as atoms, molecules, and macromolecules.

Absorption occurs when the frequency of the incoming light is equal to the
energy difference between a molecule’s ground and excited states. The
excitation of an electron from the ground state to an excited state is described
as an electronic transition (Figure 1), this is the key fundamental of molecular
spectroscopy
 Figure 1.
Absorption excitation of an electron
The energy difference of each ground/excited state pair corresponds to an
absorption band. The relationship between the energy difference and
wavelength is described by the Planck equation.

E=hν=hc/λ

where E is the energy required to promote an electron from the ground to

excited state, h is Planck’s constant, ν is the wavenumber, c is the speed of
light, and λ is the wavelength.

Planck’s equation demonstrates that the less energy needed to excite the
electrons, the longer the wavelength of the absorption band. The absorption
bands are indicative of the molecular structure of the sample and will shift in
wavelength and intensity depending on the molecular interaction and
environmental conditions. These bands are typically broad and featureless
due to the numerous molecular vibrational levels associated with the
electronic energy levels.

Figure 2.
Example absorption spectrum. The peak around 280 nm requires less
energy to promote electrons into the excited state than the peak
around 215 nm.
UV-Visible/NIR spectroscopy can be divided into ultraviolet, visible, and near-
infrared regions of the spectrum. The ultraviolet region is defined as 180 to
400 nm, visible between 400 and 800 nm, and the near-infrared is from 800 to
3200 nm.  Near-infrared light is generally poorly absorbed because its photon
energy is insufficient to induce electronic transitions and its frequency is
greater than the natural vibration frequency of most chemical bonds. 
However, since the frequency in the NIR is close to the overtone frequency of
many natural vibrations, weak substance-specific absorption bands can be
detected. It is convenient for a range of nondestructive measurements, such
as determining the moisture, sugar, lipid, protein content of foodstuffs and for
identifying medicinals.

Beer-Lambert Law
A UV-Visible/NIR spectrophotometer measures the transmittance or the
amount of light transmitted through a sample by ratioing the intensity of the
incident light (I0) to the intensity of the transmitted light (I).

T= I/I0

Figure 3. Incident light (I0)

passing through a sample that transmit light, I.
The relationship between transmittance and absorbance is described by the
following equation

abs  =   2-log⁡I/I0 ∙100       =   2-log (%T)

Absorbance measurements are frequently used to quantify an unknown

sample’s concentration by exploiting the Beer-Lambert Law that describes
how light is attenuated based on the materials it passes through.  The
transmittance, and therefore the absorbance, are directly proportional to a
sample’s concentration, c, molar absorptivity, ε , and cuvette pathlength, l.

I=I0 e-εcl
Taking the logarithm on both sides and transforming the formula,

-log I/I0 = εcl

If the left side -log(I/I0) is defined as the absorbance A, then

A= εcl

The amount of light absorbed by the sample depends on the number of

molecules interacted with. The more concentrated a sample is, the more
molecules are present and the higher the absorbance. Likewise, the longer
the pathlength of the cell, the greater the distance that the light travels through
the sample, increasing the number of molecules interacted with and therefore
the absorbance. To compare the absorbances of two solutions with either
different concentrations or pathlengths, there needs to be a constant variable
to normalize the data on. Additionally, to determine a sample’s concentration
by measuring absorbance, the cell pathlength and the strength of the
electronic transition of the chromophore must be known.  This constant or the
probability of the electronic transition occurring is the molar absorptivity. Since
molecules have different electronic transitions of varying strengths, the molar
absorptivity will vary depending on the transition being probed and is therefore
wavelength dependent.

Aside from transmission and absorption, UV-Visible spectroscopy can also

measure the reflectance of a sample, or how effective a surface is in reflecting
the total amount of incident light. Reflection occurs when light strikes a
material’s surface and causes a change in the direction of the light waves.
There are two types of reflectance: specular and diffuse, where the sum of the
two components is the total reflectance. Specular reflectance is light reflected
at same angle as the incident beam and diffuse reflectance is light reflected in
a different direction to the incident light, shown in Figure 4.

UV-Vis Spectroscopy Experimental

Choosing a Cuvette
When choosing a suitable cuvette to use for your application, we need to
consider the material of the cuvette and the volume of sample required.
Figure 18 shows the transparency of different cuvette materials. For samples
that absorb below 350 nm, quartz or UV disposable cuvettes are necessary.
However, it’s advised to compare the disposable and quartz cuvettes options.
Disposable cuvettes are made of plastics that still absorb so if the sample
absorbs strongly and a higher photometric range is required, quartz cuvettes
are a better choice. Disposable cuvettes are also not an option for the near-
infrared measurements since the material absorbs above 1000 nm.

Figure
18. Transparency of some cuvette materials for UV, visible, and NIR
regions.
Cuvettes are also broken down into macro, micro, and sub-micro volumes.
Assuming the pathlength is 1 cm, (the standard for most UV-Visible
measurements),  macro cells typically require 2.5-4 mL of sample and micro
cells require 250 to 1000 mL of sample. Here the cuvette walls are tapered to
accommodate smaller sample volumes. Sub-micro cells can hold 10 to 250
mL. However, these volumes will change with the pathlength of the cell, so
longer pathlengths require more volume than shorter pathlengths.  Micro and
sub-micro cells also have self masking options, where the cuvette walls are
black. The windows of micro and sub-micro cells are typically smaller than the
standard beam dimension to accommodate smaller sample volumes. While
the bandwidth of the instrument and therefore beam dimensions can be
reduced, any light incident on the cell walls that does not pass through the
sample can introduce stray light effects, resulting in inaccurate absorbance
values and a reduction in photometric linearity. The z-height is also another
important characteristic of cuvettes. The z-height is the height from the base
of the cell to the center of the light beam and will differ for different instrument
manufacturers. The z-height for the JASCO V-700 Series spectrophotometers
is 15 mm.
Solvent Selection

Solvents and substrates should be carefully selected for UV-Visible/NIR

experiments. If the sample is liquid, it should be soluble in the solvent
selected and assist in maintaining sample stability. Ideally, the solvent or
substrate should be transparent in the wavelength range where the sample’s
chromophore absorbs, to reduce any additional absorbance that could
potentially reach the limits of the instrument’s photometric range.

Response and Scanning Speed

The response is the amount of time that the data is integrated over or the
length of time the detector collects photons before transferring the signal to
the A/D converter for processing. The square root of the response is
proportional to the signal to noise, so the longer the response the better the
S/N. Increasing the response will have a more substantial effect when a
sample’s signal is small since there is less light throughput.

The scanning speed determines how quickly the monochromator scans

through the specified wavelength range to acquire data points at the specified
data pitch. When used in continuous scan mode, the scanning speed must be
selected with an appropriate response to prevent distortion in the measured
spectrum. The following guideline can be used when selecting the response
and scanning speed

Response × Scanning speed <  FWHM/10

where FHWM is the full width at half the peak height of the target peak.

UV-Vis Spectroscopy Applications

Analysis of the Melting Temperature and Thermodynamic Parameters of
a Nucleic Acid using a UV-Visible Spectrophotometer
This application note reports the evaluation of the melting temperature and
thermodynamic parameters of a nucleic acid sample. The absorbance at 260
nm was measured for different sample concentrations while ramping the
temperature2) using a V-700 Series UV-visible spectrophotometer with PAC-
743 Automatic 6/8-position Peltier cell changer, which can measure up to
eight samples simultaneously.

Water Analysis using a UV-Visible Spectrophotometer with a 30 cm Cell

This application note introduces a new method to analyze superficially clean
water using a UV-Visible spectrophotometer with a 30 cm pathlength cell. The
extended sample compartment is dedicated to the 30 cm cylindrical cell which
provides precise measurements of samples with extremely low absorbance
values that cannot be measured using a standard 10-100 mm path length cell.
By placing an integrating sphere in the extended compartment, all transmitted
light can be detected and integrated to achieve a maximum absorption signal.

Evaluation of UPF for Sun Protection Fabrics

This application note evaluates UPF, UPF rating, and UVA and UVB
transmittance of sun protection fiber products using a UV-Visible
spectrophotometer and the UPF calculation system. Fluorescence
measurements were also obtained to corroborate the UV-Visible results.