Fundamental Properties Of UV-Visible Spectroscopy

Fig:-1 Electronic Transition

1.    σ to σ* Transitions

• An electron in the bonding’s σ orbital is stimulated to move to its corresponding antibonding orbital.
• It requires a lot of energy because of more distance.                                                                               
• For example, methane, which only has C-H bonds and can undergo σ to σ* transitions, has an absorbance maximum at 125 nm.
  2. n to σ* Transition
• These are saturated compounds containing atoms with lone pairs, or non-bonding electrons, may undergo transitions from n to σ* Transition.
• It requires less energy than σ to σ*.
• It has an absorbance at 150-250 nm. (Sheikh Wajiha Shabbir & Shilpi Chauhan, 2024)
  3. n to π* and π to π* Transitions
• The excited state π* is reached by an electron moving from n or π. 
• It serves as the foundation for the majority of organic compound absorption spectroscopy. This is due to the spectral range that is appropriate for experimentation, which is between 200 and 700 nanometres. (Azhar S. Alaboodi et al., 2025)

2.2. Terminologies

1.  Wavelength

The distance between two successive points in the same wave phase, such as crest to crest or trough to trough, is known as the wavelength. It is commonly represented by the Greek letter λ(lambda) and indicates the length of one full wave cycle.

2.   Radiant Power

The rate at which a source emits or transfers electromagnetic energy, such as light or infrared radiation, is known as radiant power.

3.   Transmission

The percentage of incident light that flows through a sample is known as its transmittance. The ratio of the incident radiant power to the transmitted radiant power.

4.   Absorbance

The amount of light absorbed by a sample is expressed as a logarithmic measure of absorbance, formerly known as optical density.

Absorbance is directly proportional to the concentration of absorbing species and the pathlength of the sample, according to Beer Lambert’s law.

5.   Chromophores

These are the compounds which shows the presence of a functional group that gives the compound its colour or any group that exhibits absorption of electromagnetic radiation in both the visible and ultraviolet spectrums.

These are covalently unsaturated spectrums.

Ex. NO2 group

6.   Auxochromes

A group that does not act as a chromophore on its own, but when combined with one, alters absorption maxima at longer wavelengths and increases absorbance intensity. For instance, the groups -OH, -NH2, -OR, -NHR, -NR2, etc. Benzene’s absorbance maxima shifts from 255 to 280nm.(Ghosh & Nandi, n.d.)

2.3. Spectral Shifts

In UV-visible spectroscopy, a “spectral shift” is a change in the absorption maximum (either to higher or lower wavelengths) brought on by structural or environmental changes in the sample. By examining these shifts, one can gain insight into the interactions and characteristics of molecules. (Sheikh Wajiha Shabbir & Shilpi Chauhan, 2024)

Fig:-2 Spectral Shift

• Hypsochromic Shift Or Blue Shift 

Changes in the polarity of the solution or the elimination of conjugation in a system may cause a shift in absorbance maxima towards the shorter wavelength.

• Bathochromic Shift Or Red Shift

A shift in absorbance maxima toward longer wavelengths brought on by a change in solvent or the presence of particular groups like -OH or -NH2 group (auxochrome).

• Hyperchromic effect 

A possible increase in absorption intensity brought on by the addition of auxochrome. For instance, adding a methyl group to pyridine at position two results in hyperchromic effect 

• Hypochromic effect

Reduction in absorption intensity, potentially brought on by groups that have the ability to change the molecular structure. For instance, a hypochromic effect is produced when a methyl group is introduced at position 2 of the biphenyl moiety. (Ghosh & Nandi, n.d.)                                                                                                     

2.4. Beer and Lambert’s Law

The Beer-Lambert’s law is the most crucial idea in absorption analysis. According to this law, concentration and absorbance have a linear relationship for a given ideal solution as long as the pathlength remains constant; the absorptivity € is constant for every molecule at every wavelength.

                         A = €cl

Where,

A= Dimensionless quantity known absorbance

€ = Absorptivity or Molar Extinction Coefficient, also known as Epsilon

c = concentration of the absorbing species solution

l = Light’s pathlength through the solution; it is commonly expressed in centimetres

 Deviations from beer lambert’s law   

 If a graph between absorbance and concentration shows a straight line going through    origin, the system is said to be Beer and Lambert’s law. However, the linear relationship  between absorbance and concentration is never entirely maintained, especially at higher concentrations; as a result, the absorption curve varies as the solution’s concentrations does.

The deviation could be either positive or negative.

• Positive deviation occurs when the resulting curve is concave upward
• Negative deviation occurs when the resulting curve is concave downward

 The reasons for deviation of beer lambert’s law

1. Since the type of ionized species in solution changes with concentration, the law is not applicable if the substance ionize, dissociates, or associates in solution.

It is also called as chemical deviation.

One significant chemical deviation is colour change brought on by solvent dilution. Example: A deviation in the absorption curve is expected because benzoic acid and benzene combine to form a dimer.

2. If a solute forms a complex, the law does not apply; instead, the concentration determines the complexity’s composition and degree.
3. A large number of electrolytes may alter the extinction coefficient and shift the λmax.
4. Beer’s law is broken if a concentration change results in appreciable change    refractive index.
5. Deviation may also result from changes in pH brought on by variations in solute concentrations.
6. Deviations are anticipated if the monochromator’s slit width is incorrect since it may permit undesired radiation to reach the detector. (instrumental deviation). (Smith et al., 2015)

 3.Instrumentation 

Components – A spectrophotometer is a device that measures a sample                      transmittance absorbance in relation to the electromagnetic radiation’s wavelength.

To gather UV absorption spectra, two kinds of absorbance instruments are available land they are:

• Single beam uv spectrophotometer
• Double beam uv spectrophotometer

To analyze one wavelength at a time, the single beam instrument places a filter or monochromator between the source and the sample.

More accurate analysis is mad possible by the double beam instrument’s single source     and monochromator, which are followed by a splitter and a number of mirrors to direct the beam to a reference sample and the sample for analysis. Double beam UV spectrophotometers are typically faster and more effective simultaneous instruments.

The essential components of a spectrophotometer are :

1. Light source
2. Monochromators (wavelength selector)
3. Sample cell
4. Detectors
5. Readout system(Owen, n.d.)

3.1. Light Source                                                                                                               

The perfect light source would produce long term stability, low noise, and a consistent intensity across all wavelengths. Various sources of ultraviolet radiation include:

1. Hydrogen lamp
2. Tungsten halogen lamp
3. Deuterium lamp (Owen, n.d.)

1. Hydrogen Lamp  

• Hydrogen gas is kept at a comparatively high pressure in hydrogen lamps.
• The lamp will produce excited hydrogen molecules that emit ultraviolet light when an electric discharge is passed through it.
• Instead of emitting a simple hydrogen spectrum, the high pressure in a hydrogen lamp causes the hydrogen to emit a continuum.
• The lamps are widely used, steady, and stable.

2. Tungsten halogen lamp

• Filament powers the tungsten-halogen lamp.
• The filament heats up and releases light when a current flows through it.
• The lamp produces good intensity across the whole visible and near-infrared range (350nm- 3000nm) as well as a portion of the UV spectrum.
• This kind of lamp is usually lasts 10000 hours and has very little drift and nonvisible. 

3. Deuterium arc lamp

• The deuterium arc lamp produces a useful intensity in the visible range of 185 to 400 nm and a good intensity continuum in the ultraviolet region. Even though contemporary deuterium arc lamps have low signal noise, the instrument’s overall noise performance is frequently limited by lamp noise.
• A deuterium arc lamp gradually loses light intensity over time.
• A lamp of this type usually has a half-life of about 1,000 hours, which is the amount of time needed for the intensity to drop to half of its starting value. (Azhar S. Alaboodi et al., 2025)

3.2.  Monochromator (wavelength selector)

Monochrome devices, which are thought to be superior to filters in terms of efficiency, are used to transform heterochromatic or multi-coloured light into monochromatic light. 

The following parts make up the monochromator:

1. An entrance slit 
2. Dispersing device (Prism and grating)
3. An exit slit
4. Focusing lens

1. An entrance slit

Polychromatic light enters the monochromator through the entrance slit, and then the beam is collimated and angled toward the dispersion component.

2. Dispersing device

Prism – A prism uses sunlight to create a rainbow. Spectrophotometers operates on the principle of same principle. The dispersion produced by prisms is angularly nonlinear, despite their simplicity and low cost. Additionally, temperature affects the angle of dispersion.

Grating – These gadgets are constructed from glass blanks with extremely thin grooves. Reflecting source is created by applying an aluminium coating. Depending on the wavelength, light falling on the grating is reflected at various angles. Light is simultaneously dispersed and focused by a concave grating. (Owen, n.d.)

3.3. Sample Cell or Sample holder

For radiation to flow through, they require transparent containers. The cuvettes with wavelengths between 350 and 2000 nm are made up from quartz or fused silica. In the visible spectrum, these cells are likewise transparent. (Azhar S. Alaboodi et al., 2025)

3.4. Detectors

A light signal is transformed into an electrical signal by a detector. Over a broad range of low noise and high sensitivity, it bought to provide a linear response.

1. Photomultiplier tube detector
2. Barrier layer cell ( Photoholding cell) (Mandru et al., 2023)

1. Photomultiplier tube detector

The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It consists of a photoemissive cathode that releases electrons when exposed to photons of radiation and an anode that releases multiple electrons for each electron that strikes it.  A photon of radiation enters the tube and hits the cathode, causing the release of multiple electrons.  These electrons are driven by the initial anode, which is 90 volts more positive than the cathode.  Multiple electrons are released for each incident electron that hits the first electrode.

The photomultiplier tube combines multiple amplification stages inside the tube body with signal conversion. Spectral sensitivity is determined by the cathode material’s composition.  Across the whole UV-visible spectrum, a single photomultiplier produces good sensitivity.  At low light levels, this kind of detector produces high sensitivity.  High sensitivity, on the other hand, is linked to low concentrations in analytical spectroscopy applications, which lead to low absorbances and high intensity levels.  The detector needs to have low noise at high intensity levels in order to precisely detect minute variations between blank and sample measurements. Resulting in the emission of multiple electrons for every electron that is incident. In order to create more electrons

that are accelerated towards the anode, these electrons are subsequently accelerated towards the second anode. The anode is where the electrons are gathered. Each initial photon has now generated between 106 and 107 electrons. They measure and amplify the resultant current. Photomultipliers are extremely sensitive to visible and ultraviolet light. Their reaction times are quick .  (Sheikh Wajiha Shabbir & Shilpi Chauhan, 2024)

2. Barrier layer cell 

The measurement and detection of radiation in the visible spectrum is the main application for barrier layer cells.  This cell has a maximum sensitivity of roughly 550 nm. 

 It is made up of a flat copper or iron electrode with a layer of semiconducting material, like selenium copper oxide, deposited on it. The second or collector electrode is a transparent metallic layer of lead, gold, or silver that is present on the semiconductor's surface.  

The metal-selenium interface serves as a barrier to prevent electrons from passing through.

If the film is connected to the plate on the opposite side of the semiconducting layer via an external circuit and the resistance is very low, electrons from the oxide layer have enough energy to cross this barrier and move from semiconductor to metal film. The current generated by the flow of electrons can be measured with a galvanometer or microammeter.  (Sudarshan and Sweta 2023, n.d.)

3.5. Readout system 

Used for amplification of electric signal and alteration of DC to AC.

Generated electric signal is amplified and finally recorded in computerized system.

This is the main system which shows the process is completed or not by giving out the response/result in the form of spectrum, graph, numeric representation.

Without this we can do whole analysis but cannot get the result of report so to get the report of analysis this system is required.

It mainly includes a computer with proper software connected to the UV instrument and attached with printer.

4.Limitations and Challenges

Despite its versatility and widespread use, UV-Vis spectroscopy is not without limitations. Both theoretical and instrumental constraints can influence accuracy, reproducibility, and interpretive clarity. Understanding these challenges is critical for ensuring reliable analytical performance and appropriate application selection (Mandru et al., 2023; Sheikh Wajiha Shabbir & Shilpi Chauhan, 2024; Sudarshan and Sweta 2023, n.d.)