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UV-Vis Spectroscopy | Fundamentals of UV Visible Spectroscopy

UV-Vis Spectroscopy – Fundamentals of UV Visible Spectroscopy
B.Sc (USJ) (Chemistry, Environmental management, Bio-Technology), M.Sc (Industrial analytical chemistry)(Reading), HND (Software Development)
Last Updated On : November 10, 2021
Published Date : November 5, 2021
The Table of Contents show

The majority of the organic molecules and functional groups are transparent to ultraviolet (UV) and visible light. UV Visible spectroscopy (ultraviolet-visible spectroscopy) uses this electromagnetic radiation ranges to generate spectrum and gain other information about molecules. UV-Vis spectroscopy has more limited applications when compared to spectroscopic methods like infrared or NMR (H-NMR, 13C-NMR). UV Visible spectroscopy is mostly applied in analyzing organic compounds.

So, molecules have the ability to absorb those transmitted radiations from those wavelengths and use them to excite their electrons to a higher energy level from the ground state. By detecting the absorbance or the transmittance at different wavelengths, details/ information about the molecule and the solution can be gained.

For example, these data can be used to calculate the concentration of a solution or get an idea about the structure of the molecule. Also, if these results were combined with more detailed spectra like Infrared or NMR, can get very valuable information about the structure of the molecule.

How does electronic excitation work?

The excitation process
Figure 01: The excitation process

When radiation (contains a range of wavelengths) is transmitted through a transparent material/ molecule, a portion of that radiation can be absorbed by the material. If the transmitted radiation (transmitted beam) passes through a prism, a spectrum can be observed. This is called as “Absorption spectrum”. That spectrum contains a gap because the material/ molecule has absorbed a specific wavelength from the incident beam.

UV-Visible spectrometer
Figure 02: UV-Visible spectrometer

Molecules or atoms use that absorbed energy to excite their electrons to an excited state (higher energy level) from the ground state (lower energy level). The energy difference between the ground and the excited states is exactly equal to the amount of radiation absorbed.

In UV-Vis spectroscopy, radiation from ultraviolet and visible light excites electrons of the molecules from a ground state to an excited state. In this process, electrons were excited from occupied molecular orbital to unoccupied molecular orbital.

Normally the energy difference between electronic energy levels in most of the molecules is ranges from 125 kJ/mol to 650 kJ/mol.

Electronic excitation
Figure 03: Electronic excitation

Molecular orbital theory (MOT) and electronic excitation

The outermost energy level called “valance shell” is the only energy level that participates in electronic excitation and bonding. So that energy level is the only energy level considered in molecular orbital theory.

Normally, the most possible transition happens between “Highest occupied molecular orbital” (HOMO) to “Lowest unoccupied molecular orbital” (LUMO). LUMO has greater potential energy than "HOMO".

Generally, molecules contain 3 types of molecular orbitals. (Please take a look at figure 04)

  1. Bonding molecular orbitals – σ orbital, π orbitals
  2. Nonbonding orbitals - n
  3. Antibonding molecular orbitals – σ* orbital, π* orbitals

The σ orbitals correspond to σ bonds. The π orbitals correspond to π bonds. Nonbonding (n) orbitals contain unshared electron pairs.

In most of the molecules, the lowest energy occupied molecular orbitals are σ orbitals. The π molecular orbitals have much higher potential energy than σ orbitals in most cases.

σ < π < n < π* < σ*

It can be seen above; how the potential energy of the molecular orbitals changes with respect to one another. But in some special cases like N2 this pattern changes (π < σ < π* < σ*). (Because of the magnetic properties of the molecule – N2 is a diamagnetic molecule)

In molecules, there are few possible electronic transitions. They are explained in the below figures.

Electronic energy levels and transitions
Figure 04: Electronic energy levels and transitions

Transition typeλ range (nm)Examples
σ to σ*< 180Alkanes – (CH4 – λmax = 125nm)
n to σ*150 - 250H2O - λmax = 167nm
π to π*200 - 700Unsaturated functional groups
n to π*200 - 700Unsaturated functional groups
Table 01: Electronic transitions and their wavelength ranges [2]

π orbital systems of organic molecule

In, organic molecules like alkenes (ex: ethylene, 1,3-butadiene) only transition that occurs within UV – visible range is the π to π* transition. That π to π* transition can be described using an π orbital system diagram. π orbital system of 1,3-butadiene is given below.

π orbital systems of 1,3-butadiene
Figure 05: π orbital systems of 1,3-butadiene

In the above π orbital system diagram, there are 4 molecular orbitals. In this conjugated system, 4 atomic p orbitals that contribute to the π bonds are linearly overlapped to make those molecular orbitals.

When the number of nodes increases, that increases the energy of that molecular orbital. The meaning of the Node is, in the location of a node, there is a zero possibility to electron to exist.  

When radiation was absorbed, electrons excite and the transition happens to the LUMO from "HOMO".

Electronic transitions in UV-Visible spectroscopy 

In UV-Vis spectroscopy, when a molecule absorbs energy from light, it excites its electrons to an excited electronic level from the ground state.  Molecules absorb a range of wavelengths (range of energy) because molecule also contains vibrational and rotational energy levels.

Important electronic transitions
Figure 06: Important electronic transitions

Electronic transitions of organic molecules


Alkanes are saturated organic molecules. They don’t have any double bonds or triple bonds or any kind of unsaturation. Also, they don’t contain any unshared electrons. So, the only possible electronic transition that happens in alkanes is σ to σ* transition. As discussed previously, that kind of transition requires much higher energy. They absorb UV radiation at very low wavelengths that cannot even experimentally accessibly using typical spectrophotometers.

Alcohols, Ethers, Amines, and Sulfur Compounds

All these molecules are saturated molecules that contain non-bonding electron pairs. Because functional groups are saturated, and they have non-bonding electrons, the possible electronic transition is n to σ* transitions. They are also high energy transitions (low wavelengths) but they are within the experimentally accessible range of spectrophotometers.

  • Alcohols and amines – 175 to 200nm
  • Sulfides and organic triols – 200 to 220 nm

Alkenes and alkynes

Alkenes (-C=C-) and alkynes (-C≡C-) are unsaturated organic molecules that contain π bonds. So π to π* transitions become possible in these molecules. Alkene absorbs around 175 nm and alkynes absorb around 170 nm.

Also, their λmax and absorption intensity are sensitive to substitutions. (Auxochrome can increase wavelength and absorption)

Carbonyl compounds

Carbonyl compound contains both double bond (unsaturation) and non-bonding electron pairs (unshared electrons). So, they can show n to π* transitions and π to π* transitions.

Carbonyl compounds undergo n to π* transitions at 280 to 290 nm range but they are low-intensity transitions because they are forbidden transitions.

Also, carbonyl compounds undergo π to π* transitions at around the 188 nm range.

Also, λmax and absorption intensity of carbonyl compounds are sensitive to substitutions. (Auxochrome can increase wavelength and absorption)

Forbidden and allowed transitions

Transitions that have absorption maxima value (εmax) higher than 104 are called allowed transitions. Allowed transitions are generally caused by π to π* transitions.

Transitions that have absorption maxima value (εmax) less than 104 are called forbidden transitions. Forbidden transitions are generally caused by n to π* transitions.

Designation of Absorption Bands

01. K-Bands (conjugated)

These bands originated from π to π* transitions in the molecules that have a conjugated system. (-C=C-C=C-)

02. R-Bands (Radical like)

These bands originated from n to π* transitions of a single chromophore of a molecule like a carbonyl or a nitro group.

03. B-Bands (Benzenoid bands)

These bands originated from π to π* transitions happen in the aromatic or heteroaromatic molecules.

04. E-Bands (Ethylenic bands)

These bands originated from π to π* transitions happen in ethylenic bonds of aromatic or heteroaromatic molecules.

Calculating absorbance

A = log10 (I0/IT)

A = Absorbance

I0 = Light passing through reference cell (Intensity of the incident beam)

IT = Light passing through sample cell (Intensity of the transmitted beam)

A = 0 means sample (the compound that is tested) did not absorb any radiation.

A = 1 means sample has absorbed 90% of the light passing through it.

Beer-Lambert law can be used to calculate the molar absorptivity.

A = ε × C × l

ε = A / (C × l)

A = Absorbance

ε = Molar absorptivity – extinction coefficient (dm3 mol-1 cm-1)

C = Concentration of the sample solution (mol dm-3)

l = Path length (length of the sample cell) (cm)

UV-Visible spectrometer

Generally, UV Vis spectrometer consists of three main parts.

  1. Light source – Usually deuterium lamp is used for getting UV radiation and tungsten lamp is used to get wavelengths in visible region.
  2. Monochromator – Monochromator is consisting of series of slits. The purpose of the monochromator is to sort the desired wavelength and only that specific wavelength will be interacted with the sample.
  3. Detector – generally this is a photomultiplier tube. In modern spectrometer also use photodiodes as the detector. Purpose of this is to detect the light beam that passed through the sample and measure its intensity.

In instruments like double-beam spectrometer, light beam exits from the monochromator is split into two beams. One beam is called a reference beam and the other is called a sample beam. The sample beam passes through the cell that contains the actual sample that needed to be analyzed. Reference beam reference cell that only contains the solvent that was used to dissolve the sample.

Double beam spectrometer
Figure 07: Double beam spectrometer

The sample cell must be made out of material that is transparent to UV and visible regions of electromagnetic spectra. The sample cell shouldn’t be absorbing any of the light that passes through the cell. Normally transparent, clear plastic or glass cells can be used to analysis of samples using the visible range of the spectra. But those types of cells cannot use for analysis in UV regions of spectra because those types of calls absorb UV radiation. So, quartz cells are used to analyze samples using UV radiation.

The above-described instrument is only can use to analyze using only one wavelength at a time. If a complete spectrum is required (using a range of wavelengths), this kind of instrument has some difficulties because the monochromator must be mechanically rotated each time that wavelength needs to be scanned. So, these instruments are considerably slow, and a long time is required to acquire the complete spectrum.

But in modern instruments, like “Diode-array spectrophotometer” these kinds of problems have been addressed. This instrument has an array of photodiode detectors that each can scan a narrow band of transmitted spectrum. This kind of detector can record the entire spectrum at once. Also, this kind of instrument has no moving parts. So, this is a more efficient and more reliable instrument. Also, results of the analysis conducted on this machine can be directly passed to a computer and that is helpful in getting more detailed output.

UV visible Spectrophotometer
Figure 08: UV visible Spectrophotometer

UV-Visible spectra – presentation of spectra

Generally, this kind of spectrum is plotted and recorded using absorbance versus wavelength. Then data was replot using molar absorptivity (ε) or log ε on the y axis and wavelength on the x-axis.

 When analyzing a UV-Vis spectrum, clear peak absorbance or ε can be observed. The wavelength that is responsible for the top of that peak is called (wavelength of maximum absorbance) “λmax”.

That wavelength is the wavelength that is responsible for the maximum UV or visible light radiation absorbance of a molecule (its functional groups and conjugations). Molecules use energy from that specific wavelength to excite their electrons to "HOMO" to LUMO. If that was considered as the only transaction, the absorbance value to other wavelengths must be zero. So, it should be one intense sudden peak.

But when looking at UV-Vis spectra, it can be seen some absorbance at most of the wavelengths. That happens because molecules also have vibrational and rotational energy levels. Using energy provided, electrons can transmit to those levels as well.

Factors that effect on UV or visible light absorbance of a molecule


UV or visible light absorption of a molecule is happening because electrons are excited to a higher energy level from the ground state. But the nuclei of those atoms are the deciding factor of which wavelength should be absorbed. The reason for that is nuclei (charge of the nuclei) is the factor that decides how strong electrons should interact with the atom and so it will decide the energy gap between the energy levels.

So, the energy gap between energy levels and hence the wavelength of radiation that is absorbed by a molecule is a property of a group of atoms rather than electrons electron themselves. The Group of atoms that are responsible for that kind of absorption (responsible for absorption of light/ radiation) is called a “Chromophore”. Generally, the above-mentioned group of atoms that are responsible for UV or visible light absorption is a covalently unsaturated group of atoms. (-C=C-, -C≡C-, -C=O, -C≡N, N=N, NO2)

In some references, chromophores are described as the part of the molecule that is responsible for its color. But this is a partially correct statement because if the molecule (chromophore of that molecule) absorbs radiation in the visible range (400 – 800nm), only then molecule/ compound appears as colored. So, the chromophore of the molecule may or may not impact its color depending on it absorbing UV or visible light.

It is very difficult to determine how absorption will change the structure of chromophores and their behavior. So, to predict that kind of relationship, it has to be dependent on empirical (observational and experimental) working guides.

ChromophoreExampleλmax (nm)εmax (dm3 mol-1 cm-1)TransitionSolvent
-C=C-Ethylene17115 530π → π*Vapor
-C≡C-Acetylene15010 000π → π*Hexane
  1736000π → π*Vapor
-C=OAcetaldehyde16020 000n → σ*Vapor
  18010 000π → π*Vapor
  29017n → π*Hexane
 Acetone16616 000n → σ*Vapor
  188900π → π*Hexane
  27915n → π*Hexane
-COOHAcetic acid20460n → π*Water
-CONH2Acetamide1789 500π → π*Hexane
  22063n → π*Water
-COOREthyl acetate21157n → π*Ethanol
-NO2Nitromethane2015 000π → π*Methanol
  27417n → π*Methanol
-C=N-Acetoxime1905 000n → π*Water
-C≡NAcetonitrile167weakπ → π*Vapor
-N=N-Azomethane3384n → π*Ethanol
Table 02: Common chromophores and their properties [2]


Some chromophores of organic compounds contain covalently saturated substituent groups replacing their hydrogen atoms. Those attachments of chromophores change the wavelength and intensity of the absorbance of absorption maxima of chromophores. That kind of attached group is called “auxochrome”.

Generally, those auxochrome increase absorption maxima and λmax of chromophores by extending conjugations by resonance.

Example for auxochrome group – methyl (-CH3), hydroxyl (-OH), halogen, amino (-NR2), alkoxy (-OR), sulfur compounds, etc.

Effects of substituents on absorption and wavelength (Shifts in absorption position and intensity)

There are four kinds of effects/ shifts on the absorption spectrum that can be observed because of substituents and conjugations of the molecule.

Shifts in absorption position and intensity
Figure 09: Shifts in absorption position and intensity

01. Bathochromic shift – Red shift

The shift of the absorption maxima to a longer wavelength (lower energy) is called a bathochromic shift.

This shift can be occurred due to the presence of an auxochrome, increasing conjugations, or solvent effect.

02. Hypsochromic shift – Blue shift

The shift of the absorption maxima to a shorter wavelength (higher energy) is called a hypsochromic shift.

This shift can be occurred due to the removal of conjugation or change in the solvent polarity.

03. Hyperchromic effect

An increase in absorption intensity is called a hyperchromic effect.

This effect can be occurred due to the introduction of an auxochrome.

04. Hypochromic effect

A decrease in absorption intensity is called a hypochromic effect.

This effect can be occurred due to the introduction of a group that has the ability to distort the chromophore.

Effects of conjugations to UV-Vis spectra

Conjugation means that there are linked π systems present in that molecule. The meaning of that is there are adjacent double bonds. Using the below figure, this can be understood.

Examples for conjunction double bonds
Figure 10: Examples for conjunction double bonds

When conjugation increases, the energy gap between "HOMO" and LOMO decreases. That means λmax increases when conjugation increases because energy is inversely proportional to the wavelength. That means the presence of a conjugated system increases the bathochromic shift.

A comparison of the π to π energy gap in a series of polyenes
Figure 11: A comparison of the π to π energy gap in a series of polyenes

When conjugated chromophores are present that not only results in a bathochromic shift but also increases the intensity of absorption.

Moleculeλmax (nm)εmax (dm3 mol-1 cm-1)
Ethylene17515 000
1,3-butadiene21721 000
1,3,5-hexatriene25835 000
Table 03: Intensity and wavelength variation of the conjugated compounds [2]

Effects of the solvents

Choosing the right solvent for UV-vis spectroscopy is very important. Solvents are used to dissolve samples that UV vis spectrum is required. Solvent to use in UV vis spectroscopic analysis, there are a few requirements/criteria to be considered.

01. Solvent should not absorb the UV radiation in the same range of compound that spectrum is determined. So, solvents that don’t contain any conjugated systems are much suitable.

02. It should be determined that how solvent effects the fine structure of the absorption band. There are two kinds of solvents. One is polar solvents and the other is nonpolar solvents.

Nonpolar solvents do not form hydrogen bonds with the compound (solute). So, it would not effect the fine structure of the absorption band. So fine absorption band can be observed.

But polar solvents form hydrogen bonds with the compound and will form solvent-solute complexes. Then, that would affect the fine structure of the absorption band of the complex (molecule).

Polar solvent shifts π to π* transitions of polar compounds towards higher wavelength and higher absorption intensity. Also increasing the polarity of a solvent would result in shifting n to π* transitions to a shorter wavelength.

The Woodward–Fieser rules for calculating λmax

The Woodward-Fieser rules are several sets of rules that use to predict λmax (Wavelength at the absorption maxima) of a UV visible spectrum of a specific molecule/ compound.


Before describing the calculations, some specific terminologies are important to know.

1. Homoannular dienes

Conjugated double bonds should be present in the same ring. They have s-cis conformation.

2. Heteroannular dienes

Conjugated double bonds are not present in the same ring. They have s-trans conformation.

3. Exocyclic conjugated double bonds

Carbon-carbon double bond should project outside of the ring

4. Alkyl substituents and ring residues (C – Substituents)

Only ring residues and alkyl substitutions that are attached to the carbon atoms of the conjugated system are considered

Terminologies related to dienes
Figure 12: Terminologies related to dienes

 For calculating λmax in conjugated dienes and polyenes

Using this data, the position (λmax) of π → π* transition of dienes and polyenes can be estimated using Woodward–Fieser rules.

Woodward–Fieser rule for estimating the maximum wavelength of conjugated dienes and polyenes
Figure 13: Woodward–Fieser rules for estimating the maximum wavelength of conjugated dienes and polyenes [1]


01. Estimating λmax of Myrcene

Myrcene molecule
Myrcene molecule

Base value (Acyclic molecule) – 217 nm

1 C – Substituent - 5 nm

Estimated value = 222 nm

Experimental value = 225 nm

02. Estimating λmax of 1,2,3,5-Tetrahydro-5-isopropylnaphthalene


Base value (homoannular molecule) – 253 nm

1 additional conjugated double bond – 30 nm

2 exocyclic double bonds – 2*5 nm – 10nm

4 C- substituents (ring residues) – 4* 5nm – 20nm

Estimated value = 313 nm

For calculating λmax in α, β – unsaturated carbonyl compounds

Using this data, position (λmax) of π → π* transition of α, β – unsaturated carbonyl compounds can be estimated using Woodward–Fieser rules.

Woodward–Fieser rule for estimating the maximum wavelength of α, β – unsaturated carbonyl compounds
Figure 14: Woodward–Fieser rules for estimating the maximum wavelength of α, β – unsaturated carbonyl compounds [1]


01. Estimating λmax 1-acetyl-1-cyclohexene (in methanol)

1-acetyl-1-cyclohexene molecule
1-acetyl-1-cyclohexene molecule

Base value ( α, β – unsaturated ketone) = 215 nm

1 C-substituent at α (ring residue) = 10 nm

1 C-substituent at β (ring residue) = 12 nm

Solvant correction = 0

Estimated value = 237 nm

Experimental value = 232 nm

For calculating λmax in aromatic carbonyl compounds

Using this data, the position (λmax) of the “k band” of the aromatic carbonyl compounds in ethanol solvent can be estimated.

Scott rules for estimating the position of the K band of aromatic carbonyl compounds
Figure 15: Scott rules for estimating the position of the K band of aromatic carbonyl compounds [1]

Visible spectra – colors in compounds

In the electromagnetic spectrum, a wavelength range between about 400 nm to 750nm is called visible light. Humans' eye is sensitive to this range of wavelengths and can detect colors. The light that is between 400 nm wavelength region appears as violet color and light around 750nm appears as red color.

When considering the colors of the compounds, if the compound absorbs visible light, that compound will have a color. If the compound did not absorb visible light, it won’t show any color and will appear as white.

But in the compounds that absorb visible light, the color that absorbs by the compound is not the color that the compound possesses. That is because, if we shine light in visible range into a substance, that substance absorbs a certain wavelength or range of wavelengths of radiation, and the rest of the wavelengths will be reflected. So, we only can observe the reflected wavelengths and their color only. Color that is absorbed by the substance is not observable. In a transparent substance, we can only detect transmitted wavelengths from that substance.

Compounds that contain highly extended conjugated electron systems show this kind of relationship with visible light and they can have colors. Highly extended conjugated systems of those compounds have shifted their absorbance wavelengths into much higher value and into the visible range and because of that, they can appear as colored.

Absorbed colorThe wavelength of the color absorbed (nm)Observed color
Indigo blue425Yellow
Yellow550Indigo blue
Table 04: Colors in compound [1]


β-carotene (pigment of carrot)

β-carotene molecule
β-carotene molecule

 λmax =452nm | Color = orange

Cyanidin (pigment of cornflower)

Cyanidin molecule
Cyanidin molecule

λmax =545nm | Color = blue

Malachite green

Malachite green molecule
Malachite green molecule

λmax =617nm | Color = green

Analyzing ultraviolet spectrum – A general guideline

Ultraviolet spectrum doesn’t provide many details about the structure of a substance that is analyzed in UV-vis spectroscopy. The Ultraviolet spectrum is useful when you have at least a general idea about the compound that is tested.

If you combined results from the Ultraviolet spectrum with some other advanced spectroscopic method like NMR or infrared, that would become very useful. For example, then those data can be used for purposes like identifying carbonyl groups, double bonds, aromatic rings, and other important chromophores.

Following observations can be used as guidelines when it comes to analyzing the Ultraviolet spectrum.

  • If there is a low to medium intensity (100 to 10000) single band below 220 nm wavelength range in the ultraviolet spectrum, that usually indicates n to σ* transition. That can happen because of presence of amines, alcohols, ethers or thiols.
  • If there is a low intensity (10 to 100) single band at 250 nm to 360 nm wavelength range in the ultraviolet spectrum and with no considerable absorption at shorter wavelengths (200 to 250 nm) in the ultraviolet spectrum , that usually indicates n to π* transitions. This could indicate presence of simple, unconjugated chromophores contains O, N or S atoms.
  • If there are two bands that show medium intensity (1000 to 10000) at wavelength above 200 nm in the ultraviolet spectrum, that could indicate the presence of an aromatic system.
  • If there are bands that have high intensity (10000 to 20000) at wavelength above 210 nm in the UV spectrum, that generally indicate the presence of groups like α, β- unsaturated ketones, dienes or polyenes.
  • Because compounds like simple carboxylic acids, simple ketones, amides, esters contain both π electron system and unshared electron pairs they can show two absorption peaks. One for n to π* (longer wavelength above 300nm and low intensity) and one for π to π* (shorter wavelength below 250nm and high intensity) transitions.
  • Compound that exhibits color (have absorption in visible range), may have contains extended conjugated systems or polycyclic aromatic chromophores. But also, simple nitro, azo, nitroso like compounds can exhibit color.

Sample Ultraviolet spectra

Reference UV Vis Spectra of alkenes
Figure 16: Reference UV Vis Spectra of alkenes [1]

Reference UV Vis Spectra of aromatic compounds
Figure 17: Reference UV Vis Spectra of aromatic compounds [1]

Ultraviolet spectra of pyridine and quinoline
Figure 18: Ultraviolet spectrum of pyridine and Ultraviolet spectrum of quinoline [2]

References and Attributes

[1]Tables of Spectral Data for Structure Determination of Organic Compounds. 4th ed. - Pretsch, E., Biemann, K., Clerc, T., Seibl, J., Simon, W. and Pretsch, E., 2009.
[2]Introduction to spectroscopy. 5th ed - Pavia, D., Lampman, G., Kriz, G., Vyvyan, J. and Lin, T., 2015.
Myrcene - National Center for Biotechnology Information (2021). PubChem Compound Summary for CID 31253
1,2,3,5-Tetrahydro-5-isopropylnaphthalene - National Center for Biotechnology Information (2021). PubChem Compound Summary for CID 101316547


The cover image was designed by using an image from Matylda Sękpl.wiki: Cygaretkacommons: Cygaretka, CC BY-SA 3.0, via Wikimedia Commons
Figure 08: Image by Viv Rolfe, CC BY-SA 4.0, via Wikimedia Commons
Figure 18: Image of the spectra is based on data in Ultraviolet Spectra of Aromatic Compounds, John Wiley and Sons, New York, 1951

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