Principle, working and applications of UV spectroscopy

UV spectroscopy is an important tool in analytical chemistry. The other name of UV (Ultra-Violet) spectroscopy is Electronic spectroscopy as it involves the promotion of the electrons from the ground state to the higher energy or excited state. In this article I will explain the basic principle, working and all the applications of UV spectroscopy.

Introduction to UV spectroscopy

UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet region (200-400 nm.) is absorbed by the molecule. Absorption of the ultra-violet radiations results in the excitation of the electrons from the ground state to higher energy state. The energy of the ultra-violet radiation that are absorbed is equal to the energy difference between the ground state and higher energy states (deltaE = hf).
amanGenerally, the most favoured transition is from the highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO). For most of the molecules, the lowest energy occupied molecular orbitals are s orbital, which correspond to sigma bonds. The p orbitals are at somewhat higher energy levels, the orbitals (nonbonding orbitals) with unshared paired of electrons lie at higher energy levels. The unoccupied or antibonding orbitals (pie* and sigma*) are the highest energy occupied orbitals.
In all the compounds (other than alkanes), the electrons undergo various transitions. Some of the important transitions with increasing energies are: nonbonding to pie*, nonbonding to sigma*, pie to pie*, sigma to pie* and sigma to sigma*.

Principle of UV spectroscopy

UV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution.
The expression of Beer-Lambert law is-
A = log (I0/I) = Ecl
Where, A = absorbance
I0 = intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity

From the Beer-Lambert law it is clear that greater the number of molecules capable of absorbing light of a given wavelength, the greater the extent of light absorption. This is the basic principle of UV spectroscopy.

Instrumentation and working of UV spectroscopy

Instrumentation and working of the UV spectrometers can be studied simultaneously. Most of the modern UV spectrometers consist of the following parts-

Light Source- Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.

Monochromator- Monochromators generally composed of prisms and slits. The most of the spectrophotometers are double beam spectrophotometers. The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism.

Sample and reference cells- One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV region.

Detector- Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.

Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servometer. Generally current generated in the photocells is of very low intensity, the main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals.

Recording devices- Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound.

Concept of Chromophore and Auxochrome in the UV spectroscopy

Chromophore- Chromophore is defined as any isolated covalently bonded group that shows a characteristic absorption in the ultraviolet or visible region (200-800 nm). Chromophores can be divided into two groups-
a) Chromophores which contain p electrons and which undergo pie to pie* transitions. Ethylenes and acetylenes are the example of such chromophores.
b) Chromophores which contain both p and nonbonding electrons. They undergo two types of transitions; pie to pie* and nonbonding to pie*. Carbonyl, nitriles, azo compounds, nitro compounds etc. are the example of such chromophores.

Auxochromes- An auxochrome can be defined as any group which does not itself act as a chromophore but whose presence brings about a shift of the absorption band towards the longer wavelength of the spectrum. –OH,-OR,-NH2,-NHR, -SH etc. are the examples of auxochromic groups.

Absorption and intensity shifts in the UV spectroscopy

There are four types of shifts observed in the UV spectroscopy-

a) Bathochromic effect- This type of shift is also known as red shift. Bathochromic shift is an effect by virtue of which the absorption maximum is shifted towards the longer wavelength due to the presence of an auxochrome or change in solvents.
The nonbonding to pie* transition of carbonyl compounds observes bathochromic or red shift.

b) Hypsochromic shift- This effect is also known as blue shift. Hypsochromic shift is an effect by virtue of which absorption maximum is shifted towards the shorter wavelength. Generally it is caused due to the removal of conjugation or by changing the polarity of the solvents.

c) Hyperchromic effect- Hyperchromic shift is an effect by virtue of which absorption maximum increases. The introduction of an auxochrome in the compound generally results in the hyperchromic effect.

d) Hypochromic effect- Hyperchromic effect is defined as the effect by virtue of intensity of absorption maximum decreases. Hyperchromic effect occurs due to the distortion of the geometry of the molecule with an introduction of new group.

Applications of UV spectroscopy

1. Detection of functional groups- UV spectroscopy is used to detect the presence or absence of chromophore in the compound. This is technique is not useful for the detection of chromophore in complex compounds. The absence of a band at a particular band can be seen as an evidence for the absence of a particular group. If the spectrum of a compound comes out to be transparent above 200 nm than it confirms the absence of –
a) Conjugation b) A carbonyl group c) Benzene or aromatic compound d) Bromo or iodo atoms.

2. Detection of extent of conjugation- The extent of conjugation in the polyenes can be detected with the help of UV spectroscopy. With the increase in double bonds the absorption shifts towards the longer wavelength. If the double bond is increased by 8 in the polyenes then that polyene appears visible to the human eye as the absorption comes in the visible region.

3. Identification of an unknown compound- An unknown compound can be identified with the help of UV spectroscopy. The spectrum of unknown compound is compared with the spectrum of a reference compound and if both the spectrums coincide then it confirms the identification of the unknown substance.

4. Determination of configurations of geometrical isomers- It is observed that cis-alkenes absorb at different wavelength than the trans-alkenes. The two isomers can be distinguished with each other when one of the isomers has non-coplanar structure due to steric hindrances. The cis-isomer suffers distortion and absorbs at lower wavelength as compared to trans-isomer.

5. Determination of the purity of a substance- Purity of a substance can also be determined with the help of UV spectroscopy. The absorption of the sample solution is compared with the absorption of the reference solution. The intensity of the absorption can be used for the relative calculation of the purity of the sample substance.


Guest Author: laso22 Jul 2014

I really love your work because its straight to the point and well summarized.
I would also love a detailed report on qualitative analysis of analyze using UV and properties of a sample that can be directly determined by UV. When you have a sample not fulfilling requirements in order to be analyzed using UV what should be done to it in order to be analyzed?


Guest Author: jyothi30 Aug 2014

add some more applications with examples

Guest Author: gurinder kaur15 Sep 2014

It is really good.

Guest Author: vishal shirale25 Sep 2014

please add Electronic transition with some example

Guest Author: amber17 Nov 2014

You are really stead forward man. I really like it because it was to the point which I really like. .

Guest Author: vikas05 Dec 2014

Its application is useful for me for the preparation of my final examination of chemical engineering.

Guest Author: Kumara Vyas Malliset11 Dec 2014

Its very useful for all the analysts. Explanation is very good with simple language. Really, I like it.

Guest Author: payal12 Dec 2014

it is really a very good and easily understandable information!

Guest Author: nishanth01 Jan 2015

Awesome sir. I liked the way you briefed it but if you add a more note on detectors that would be more perfect

Guest Author: M zubair31 Jan 2015

thanks sir
I like your topic because it is not comprehensive and is from point to point

Guest Author: Rajesh06 Feb 2015

Thank you so much for sharing UV principles and applications. Kindly share the working procedure with block diagram. It will be very helpful if you share other instruments principles also.

Author: Ali11 Mar 2015 Member Level: Bronze   Points : 0

It is really very nice .thank you very much

Guest Author: Mahesh05 Apr 2015

I like your article because you have given a perfect pattern of UV spectroscopy.

Guest Author: Peter20 Apr 2015

Simply precise piece of work! Well done!

Guest Author: Pruthvisinh22 May 2015

Really very good.

Guest Author: snehal D wani12 Jun 2015

It is really good all points

Guest Author: Suresh patil19 Jun 2015

Really a very good and easily understandable information!

Guest Author: Egadu Samuel15 Jul 2015

Brief but very factual scientific information.

Guest Author: Rahavi12 Aug 2015

Interesting to understand the actual concept.

Guest Author: HIRPO16 Oct 2015

Very good ideas but add also more explanation of model of UV and from what does the given UV made metals, nonmetal or etc more it is very good I can say.


Guest Author: narendra S.Tomar26 Oct 2015

Mr.Aman, thank you very much for your effort. Your content is very reliable and precise from the point of view of an academic. It is really helpful to all analytical chemists. I would like to suggest you to please add more practical and application content to the article.

Guest Author: Mihir rathod18 Jun 2016

thank you sir for this idea . it is very helpfull for me bcoz its give me most usefull infomation of uv in a small time.
kindly share working procedure with block diagram whereby that are more suitable for study.
once again thanku

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