UV-visible spectroscopy is a fast analytical technique that measures the absorbance or transmittance of light. Although the UV wavelength ranges from 100–380 nm and the visible component goes up to 800 nm, most of the spectrophotometers have a working wavelength range between 200–1100 nm.
The practical range for UV-vis spectroscopy varies from 200–800 nm; above 800 nm is infrared, while below 200 nm is known as vacuum UV. The ability of matter to absorb and to emit light is what defines its color and the human eye is capable of differentiating up to 10 million unique colors. Light passes through media (transmission), reflects off both opaque and transparent surfaces, and is refracted by crystals. Covalently unsaturated compounds with electronic transition energy differences equivalent to the energy of the UV-visible light absorb at specific wavelengths. These compounds are known as chromophores and are responsible for their color. Covalently saturated groups that do not absorb UV-visible electromagnetic radiation but affect the absorption of chromophore groups are called auxochromes. When UV-vis radiation hits chromophores, electrons in the ground state jump to an excited state, which we refer to as electron-excitation, while auxochromes are electron-donating and have the capacity to affect the color of choromophores while they do not change color themselves. Water and alcohols are mostly transparent and do not absorb in the UV-vis range and so are excellent mediums for UV-visible spectroscopy. Acetone and dimethylformamide (DMF) are good solvents for compounds insoluble in water and alcohol, but they absorb light below 320 and 275 nm, respectively, so are appropriate only above these cut-off wavelengths.
3. INTRODUCTION
UV-visible spectroscopy is a fast analytical technique that measures the absorbance or transmittance of light.
Although the UV wavelength ranges from 100–380 nm and the visible component goes up to 800 nm, most of the
spectrophotometers have a working wavelength range between 200–1100 nm.
The practical range for UV-vis spectroscopy varies from 200–800 nm; above 800 nm is infrared, while below 200
nm is known as vacuum UV.
The ability of matter to absorb and to emit light is what defines its color and the human eye is capable of
differentiating up to 10 million unique colors.
Light passes through media (transmission), reflects off both opaque and transparent surfaces, and is refracted by
crystals.
Covalently unsaturated compounds with electronic transition energy differences equivalent to the energy of the UV-
visible light absorb at specific wavelengths. These compounds are known as chromophores and are responsible for
their color.
4. Covalently saturated groups that do not absorb UV-visible electromagnetic radiation but affect the absorption of
chromophore groups are called auxochromes.
When UV-vis radiation hits chromophores, electrons in the ground state jump to an excited state, which we refer
to as electron-excitation, while auxochromes are electron-donating and have the capacity to affect the color of
choromophores while they do not change color themselves.
Water and alcohols are mostly transparent and do not absorb in the UV-vis range and so are excellent mediums
for UV-visible spectroscopy.
Acetone and dimethylformamide (DMF) are good solvents for compounds insoluble in water and alcohol, but
they absorb light below 320 and 275 nm, respectively, so are appropriate only above these cut-off wavelengths.
5.
6.
7. DESCRIPTION
UV-vis spectrophotometers direct a light source through a sample and a detector on the opposite side records
transmitted light (Figure 1).
Typically, graphs of the data have the baseline at the bottom with the peaks pointing upward and they report
wavelength in nanometers (nm) on the x-axis and absorbance (OD/A) on the y-axis (no units).
The transmittance represents how much light is absorbed at each wavelength and we are most interested in the
highest peak (Amax).
Chromophores have characteristic absorbance bands.
Changing their environment either by adding another compound or altering the physical environment, like raising
temperature, may change their energy levels and thus the wavelength and the intensity of absorbance.
8. Figure 1 UV-vis spectrophotometer schematic for a double beam instrument
9. Band shifts to longer wavelengths (red-shift) are bathochromic shifts and shifts to shorter wavelengths
(blueshift) are hypsochromic shifts.
An increase in the peak intensity of an absorption band is hyperchromism and a decrease is hypochromism.
A
10. Solvents may influence the absorbance intensity and wavelength shifts.
Figure 2 illustrates differences induced due to solubility and the interaction of chromophores and auxochromes groups of the
solvents with the methylene blue light absorbent group.
High performance liquid chromatography integrates UV-visible spectroscopy with fixed or variable incident wave length
detectors.
11. Figure 2. Methylene blue absorbance maximum intensity and its wavelength shifts induced by environmental
changes. Absorbance recorded in water, acetone, chloroform, DMSO, DMF, acetonitrile, and isopropyl alcohol.
12. In diffuse reflectance spectroscopy (DRS) a solid (usually a powder) is placed in an integrating sphere that allows adding
multiple reflections.
The reflected beam intensity, R, is then compared with a reference that gives
R∞ = R sample/R standard
which is an approximation of an infinitely thick sample.
THEORY AND PRINCIPLE
UV-visible spectroscopy is based on electronic transitions of organic molecules absorbing light that excite electrons from a lower
energy orbital (highest occupied molecular orbital (HOMO) to a higher energy unoccupied orbital (lowest unoccupied molecular
orbital (LUMO).
The energy of the light wavelength absorbed must be equal to delta Epsinon (E)of the HOMO-LUMO energy gap (Figure 3).
For conjugated pie− pie systems, the energy gap from the lower energy to the higher energy molecular orbital is smaller than isolated
double bonds, so longer wavelengths are absorbed.
For larger conjugated pie systems, the correspondingly light wavelength becomes longer too.
Light absorbance, (A), is proportional to the path length through the sample, (b), concentration, (C), and an molar absorptivity,
Epsinon (E) , that is characteristic for every compound—the Beer-Lambert law:
A = log10 Io/ I = E · b · C………………………..(1)
13. Beer-Lambert law states that
“When a beam of monochromatic light passes through a medium, the intensity of light decreases with the
increase in thickness of the medium and is directly proportional to the concentration of solute in the solution.”
14. Figure 3 Molecular orbitals and the energy gap needed to excite the electron energy state
15. The light intensity, I, is measured with respect to a reference, Io.
Rearranging Equation (1), we find the concentration as a function of the known parameters after calibration,
C = 1/· b ( A)…………………(2)
In DRS, the reflectance data are usually treated with the Schuster-Kubelka-Munk function:
F(R∞) = (1 − R∞) 2 / 2R∞ = K / S………(3)
where the numerator represents the light intensity reflected from the sample, the denominator is the light intensity
from the reference, and K and S are the Schuster-Kubelka-Munk absorption and scattering coefficients.
With Equation (3), we estimate the band gap (Eg ) of semi conductors with a linear plot of [F(R∞)hv] 2 versus hv
[F(R∞)hv] 2 = C2(hv − Eg )....................(4)
16. BEER LAMBERT’S DERIVATION
Mathematically it can be expressed as - 𝑑𝐼/𝑑𝑥 ∝ 𝐼……………..(1)
Where dI is a small decrease in intensity of light upon passing through a small distance
dx and I is the intensity of the monochromatic light just before entering the medium.
Equation (1) may be written as - 𝑑𝐼/𝑑𝑥 = 𝑎𝐼…………………(2)
Where - 𝑑𝐼/𝑑𝑥 is the rate of decrease of intensity with thickness dx , a is called the absorption co-efficient.
Integration of equation (2) after rearrangement gives, - ln I = ax+C --- --- --- --- --- --- (3)
Where C is a constant of integration.
At x=0, I=Io.
So, C = - ln Io.
Introducing this in equation (3) we get,
ln I/Io = - ax…………………(4)
Equation (4) can also be written as, I = Io 𝑒 −𝑎𝑥…………………..(5)
17. Equation (5) can also be written as, log I/Io = − a/2.303 x………….(6)
or, log I/Io = -a` x………….(7)
Where a` (= a/2.303 ) is called extinction co-efficient and -ln I/Io is termed absorbance of the medium.
Absorbance is represented by A.
Lambert’s law was extended by Beer who showed that when light passes through a solution of a given thickness,
the fraction of incident light absorbed is dependent not only on the intensity (I) of light but also on the
concentration (c) of the solution.
This is known as the Beer’s law. - 𝑑𝐼/𝑑𝑥 ∝ 𝑐…………….(8)
The two laws may be combined to write - 𝑑𝐼/𝑑𝑥 ∝ 𝐼 × 𝑐
Or
- 𝑑𝐼/𝑑𝑥 = 𝑏 × 𝐼 × 𝑐……………..(9)
When the concentration, c, is expressed in mol /L,
b is called the molar absorption co-efficient.
18. As in the case of Lambert’s law equation (9) may be transformed into,
log I/Io = − 𝑏/2.303 × 𝑐 × 𝑥…………….(10)
log I/Io = - ∈× 𝑐 × 𝑥………………(11)
Where ∈ (= 𝑏/2.303 ) is called the molar extinction co-efficient which is expressed in L/mol/cm.
The molar extinction co-efficient ∈, is dependent on the nature of the absorbing solute as well as on the wave length
of the incident light used.
The expression (equation 11) is commonly known as Beer-Lambert’s law.
19. PROCEDURE
1. Calibrate the Spectrometer
Turn on the UV-Vis spectrometer and allow the lamps to warm up for an appropriate period of time (around 20
min) to stabilize them.
Fill a cuvette with the solvent for the sample and make sure the outside is clean. This will serve as a blank and
help account for light losses due to scattering or absorption by the solvent.
Place the cuvette in the spectrometer. Make sure to align the cuvette properly, as often the cuvette has two sides,
which are meant for handling (may be grooved) and are not meant to shine light through.
Take a reading for the blank. The absorbance should be minimal, but any absorbance should be subtracted out
from future samples. Some instruments might store the blank data and perform the subtraction automatically.
20. 2. Perform an Absorbance Spectrum
Fill the cuvette with the sample. To make sure the transfer is quantitative, rinse the cuvette twice with the sample
and then fill it about ¾ full. Make sure the outside is clean of any fingerprints, etc.
Place the cuvette in the spectrometer in the correct direction.
Cover the cuvette to prevent any ambient light.
Collect an absorbance spectrum by allowing the instrument to scan through different wavelengths and collect the
absorbance. The wavelength range can be set with information about the specific sample, but a range of 200–800
nm is standard. A diode-array instrument is able to collect an entire absorbance spectrum in one run.
From the collected absorbance spectrum, determine the absorbance maximum (λmax). Repeat the collection of
spectra to get an estimate of error in λmax.
To make a calibration curve, collect the UV-Vis spectrum of a variety of different concentration samples.
Spectrometers are often limited in linear range and will not be able to measure an absorbance value greater than
1.5. If the absorbance values for the sample are outside the instrument's linear range, dilute the sample to get the
values within the linear range.
21. APPLICATIONS
In 2016 and 2017 WoS Core Collection indexed 22 000 articles that referenced UV-vis spectroscopy. We compiled the
10 000 most cited papers mentioning the technique and generated a bibliometric map of the top 100 keywords among
the articles (Figure 4).
The VOS Viewer program recognized four domains: photo catalysis, water treatment: nanoparticles and nano stucutres
crystals, complexes, and derivatives; and Ag and Au nanoparticles and antibacterial activity (beige).
Chemical engineering applications include waste-water treatment, dye degradation, characterizing colloidal
nanoparticles silver, gold, and copper metallic coordination of molybdenum and cobalt, bimetallic mesoporous
materials with cobalt and chromium, and composites of graphene oxide and silver nanoparticles.
Engineers also characterize polymer impregnation, measure the size distribution of emulsions, release rates control of
organic compounds and encapsulation and release rates of antibiotics.
UV-vis spectroscopy is a powerful tool to calculate the reaction rates. Coupled with HPLC and mass spectrometry, it
monitors several wavelengths concurrently.
UV-vis spectroscopy assesses mixing in reactors and differentiates pure from blended beverages as a quality control.
22. Figure 4. UV-vis spectroscopy correlation map using VOS viewer program.
23. In chemical engineering applications, it quantifies concentration with calibration curves and Beer-Lambert’s
equation, or evaluates shifts in the spectra from nanoparticles colloids to estimate the size distribution and the
form of the particles.
UV-vis spectroscopy is one of the most reliable techniques for nanoparticle colloidal analysis even at the
industrial scale.
In addition to determining semi-conductor band gap easily, DRS also resolves the coordination state of oxides
and especially supported oxides.
24. The biblometric map identifies nanoparticles (NPs) as the most cited keyword in the field of UV-vis (Figure 4),
which is surprising since most applications relate to measuring composition.
Gold nanoparticle (AuNPs) surfaces are easy to functionalize and conjugate with bio substrates.
Moreover, they are chemically stable, biocompatible, and support surface plasmons and are applied in optical,
electrical, chemical, and biological systems.
UV-vis measures their concentration and particle size in real time and relies on the principles of Mie theory,
coupled with the Ganns Fitting model.
AuNP’s UV-vis spectrum contains a band that varies in the range from 520–580 nm due to surface plasmon
resonance and an absorption edge at shorter wavelengths. As the nanoparticles grow and agglomerate, the red
band shifts to higher wavelengths and the accuracy is within 6 %.
The resonance band shift of silver nanoparticles (AgNPs) increases linearly (R2 = 0.99) from 397–427 nm as the
particle diameter increases from 18.2–58 nm.
25. LIMITATIONS
1. Uncertainty
Although most of the spectrophotometers have the working wavelength range from 200–1100 nm and the
spectral range corresponding to the UV-vis light is from 100–800 nm, a narrower interval from 220–780 nm
should be considered during analysis for better accuracy and reliability.
Solvents interact, such as polar molecules in polar solvents, and solute-solvent interactions are appreciable and
decrease the resolution of the spectrum.
In the case of non-polar compounds in nonpolar solvents, the effect of the solvent is negligible.
In some cases, byproducts from reactions involving the solvent interfere in the region of interest from the spectra.
Mixtures are harder to quantify, but they can be analyzed with a multicomponent analysis (MCA) method, which
assumes that all possible components forming the mixture are known and Beer Lamberts law applies for all
components.
26. 2. Sources of Error
The largest sources of error in spectrophotometry are related to sample preparation, cell handling, and
cleanliness.
The cell is a part of the spectrophotometer’s optical system and its composition and geometry influence the
precision. Experiments must be conducted in the same cell.
At high concentrations, compounds deviate from Beer-Lambert’s law, due to dimerization, decomposition,
aggregation, and precipitation.
Figure 5 compares the predicted absorbance of indocyanine green dye (ICG) in methanol with experimental
measurements.
At concentrations below 1 mol · L−1 the signal is linear, but it deviates thereafter.
Although the linear range is higher for some compounds, we recommend maintaining concentrations below 1
mol · L−1 to ensure reproducible and accurate measures.
Samples can be sensitive to dissolved gases in the solvent (like oxygen) as well.
27. Figure 5 Theoretical and measured UV-vis plot (concentration versus absorbance) of indocyanine green dye in
methanol.
28. To maximize signal sensitivity and minimize operator error, we propose a standardized methodology including
sample preparation, checking absorbance, verifying linearity, and identifying optimal dilution factor (highest
value in the linear range, Figure 6).
Increasing temperature dilates solvents and so to measure concentration accurately requires a correction factor for
the coefficient of thermal expansion.
However, volume expansion may impact absorbance less than the effect of temperature on hydration of the
chromophore compounds.
Varying pH may affect the chemical equilibrium and modify molecular and electronic structural properties, which
shifts the absorbance spectra.
30. Some compounds (molecular probes) interact at the molecular scale, which also shifts the absorbance.
Increasing concentration increases the interaction frequency, which may form dimers.
High concentrations improve sensitivity at the risk of deviating from the linear region of the absorbance curve
(Beer Lambert's law).
Compounds with high molar absorptivity (E) are more sensitive (at the same concentration) than compounds with
a lower (E), which, coincidentally, will minimize molecular interactions between the compounds and impurities.
The monochromator chooses the wavelength of light that passes through the exit slit bandpass (Figure 1)and this
slit width can yield some deviation. Stray light, from instrument malfunction, diffraction, or light scattering,
decreases the measurable absorbance range and alters the relationship between concentration and absorbance.
It includes heterochromatic electromagnetic radiation outside the bandpass of the monochromator
31. DETECTION LIMITS
Instruments estimate noise through the reference sample.
Noise is the major limiting factor that affects precision at low absorbance.
Ideally, spectrophotometer analysis should measure noise at all wavelengths, which is time consuming.
Practically, we analyze the base line flatness of noise and compare it to the sample measurement, and it also
reveals instrumental problems resulting from light source exchanges or switching filters.
For measurements, the instrumental resolution should be at least 10 times as high as the natural bandwidth of the
peak measured.
The absorbance value will be lower than the true value if the instrumental resolution is insufficient.
Figure 7 displays the signal to noise relationship.
The absorbance ratio of the sample versus noise should be greater than 2 and ideally 3 to have a valid
measurement.
32. Figure 7. Signal-to-noise ratio plot for UV-vis measurements. The arrows show the difference of the sample’s
and noise absorbance intensity.
33. CONCLUSIONS
UV-vis spectroscopy is one of the simplest and most effective analytical techniques to measure species
concentration in the liquid phase.
It is an effective tool to assess reaction kinetics and is applied widely by research and industrial laboratories for
both quantitative and qualitative analysis.
The Beer-Lambert law adequately characterizes the relationship between absorbance and concentration for most
compounds up to A = 1, without a calibration curve.
UV-vis diffuse reflectance spectroscopy measures the semiconductor band gap, which is especially useful in
photocatalysis research.
It also allows for characterizing the coordination state of oxides and supported oxides, which is key information
in the development of oxide catalysts.
34. Over 22 000 articles applied this technique in 2016 and 2017 and the most frequent keywords in the articles were
nanoparticles, TiO2, and degradation.
The top journals include RSC Advances with 441 articles in the top 10 000 cited articles, followed by Journal Of
Materials Science-Materials In Electronics , Applied Surface Science , and Journal Of Molecular Structure.
As a category, WoS assigned 860 of the top 10 000 to chemical engineering, which ranks it 6th behind physical
chemistry, multidisciplinary materials science, multidisciplinary chemistry, applied physics, and condensed matter
physics.
35. REFERENCES
Experimental Methods in Chemical Engineering: Ultraviolet Visible Spectroscopy—UV-Vis Fellipy S. Rocha,etal
The Canadian journal of chemical engineering, volume 96, December 2018
