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UV Vis Spectroscopy

The electromagnetic radiation in the ultra violet and visible region of the spectrum (40 to 1000 THz) is found to be higher in frequency as compared to either the microwave or infrared and hence makes a very good energy level spacing association for large transitions between electronic energy levels.

The UV VIS spectroscopy can help us study the molecules around us in any type of phase but most preferred is for liquid samples. In recent times this UV VIS spectroscopy has brought in some interesting techniques, instruments and data processing methodology which ultimately helped in getting better and crisper software. The practical application of this UV VIS spectroscopy also was visible in all kinds of chemo-metric techniques.


UV VIS Spectroscopy Definition

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This UV VIS spectroscopy is based on the Bohr Einstein frequency relationship of ∆ E = E2 – E1 = hv 

This relationship helps in getting the discrete atomic or molecular energy states E1 with frequency v of the electromagnetic radiation. The proportional constant h is Planck’s constant (6.626 x 10-27 erg s). In spectroscopy it is appropriate to use the wave number Ṽ instead of frequency v. 

∆ E = E2 – E1 = h c Ṽ 

Where, v = c / λ = c Ṽ

The absorbed or emitted radiation of frequency or wave number can be assigned to specific energy difference. 

Hence, Ṽ = ∆ E / h c = E2 / h c – E1 / h c = T2 – T1

Absorption spectroscopy in the ultraviolet and visible region can be characterised with the above mentioned relationship. Within the overall range of electromagnetic radiation which is of interest to lot of chemists and both UV and VIS absorption spectroscopy covers a specific narrow frequency only or wave number region. 

Nevertheless this range gives us an important message that since energy differences correspond to those of electronic states of atoms and molecules which corresponds to electronic spectroscopy. Moreover, the visible spectral region in the interactions between matter and electromagnetic radiation combines themselves as colour. 

The short wave length limits is restricted by appratus and by other experimental techniques. The long wave length limit depends less on considerations of appratus as apart from a few exceptions, most of the compounds exhibit no absorption which are traceable to the electronic excitation in this region. 
  • The vibrational quanta of the excited state can be observed in the absorption spectrum and compared to this the ground state in fluorescence and phosphorescence spectra.
  • Very often the fluorescence spectrum is approximately a carbon copy of the absorption spectrum
  • Due to low account energy level of the triple state T1, the phosphorescence spectrum is displaced strongly towards the red region state as the fluorescence and phosphorescence spectra are normally separated clearly.
There are various types of UV VIS spectrometers, with the specific one with a double beam spectrophotometer having a UV visible light source, along with two cells through which the light is allowed to pass and along with that a detector which helps in measuring the amount of light passing through the cells. There are basic types of spectrometers which helps in measuring the absorbance limits of specific wave length or can scan the entire range of light arrays.

New types of spectrometers managed by computers which are then controlled through specific instruments and instructions to allow greater accuracy and flexibility. This also help in overlaying the spectra of a reagent mix checked with a specific time frame or can also help in getting a perfect calibrating graph in order to find the unknown reagents concentration. The single beam UV VIS spectroscopy work on the same general principles but measure the absorption of the reference first and then follow up with the sample.

These instruments can scan UV VIS spectroscopy range or can be utilised for even single form wavelength. The DAD or the diode array detector helps in getting detections simultaneously for an entire range of material, which helps in faster quantification of the absorbing material. The UV VIS spectroscopy refers to the radiation range of wavelength 200 to 800 nm. At the same time there are many matter which absorbs lower than 200 nm but this spectrum is very difficult to examine unless these are recorded in a vacuum. 

In case of double beam UV VIS spectroscopy the light is allowed to be split into two parallel beams. Each of these are allowed to go through a cell. 
One of these cells contain the sample while the other one contains only the solvent part. The detector helps in getting the light intensity measured by transmittance of the solvent alone (I0) and then this is compared to specific light intensity of light allowed to transmit through sample cell (I). Finally the absorbance A is calculated from the equation given out as:
A = log10 [I0 / I] 

UV VIS Spectroscopy Applications

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The UV VIS spectroscopy is a type of absorption spectroscopy in which the ultra violet region is absorbed by the molecule. Absorption of the ultra violet radiations result in the electron excitement which helps these to move from ground state to higher state. 

The amount of energy specific to ultra violet radiation which are absorbed is found to be equal to the difference in energy existing between the ground state and the higher energy state. This is given out as 

∆ E = hf
  • The UV VIS spectroscopy helps in detecting the presence of type of chromophore in a compound or even the absence of it. They can help in detecting the absence of conjugation, carbonyl group, benzene or aromatic compound or even bromo / iodo atoms
  • The limits of conjugation can also be detected in the poly – enes with the help of this UV VIS spectroscopy and with the increase in double bonds the absorption shifts towards the longer wavelength.
  • The UV VIS spectroscopy can also help in identifying unknown compounds. The spectrum of a known compound is compared to a referred spectra of an unknown compound and the identification is carried out for specific match.
  • The configuration of various geometrical isomers are also carried out with the help of UV VIS spectroscopy by observing the cis and trans forms of alkenes as both absorb different wavelength of light.
  • The purity of the substances is also identified with the help of the utilised UV VIS spectroscopy. The sample for absorption is compared and matched for with the reference solution and the intensity of the absorption of this is used for identifying the purity level of sample substance.

UV VIS Spectroscopy Instrumentation

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The instrumentation or characterization, as it is better known as, techniques like UV visible spectroscopy are usually carried out by utilizing instruments like Hitachi U 3310 UV VIS spectrophotometer. The characterization is basically used for understanding the spectral lines of a given material.

The atomic spectroscopy always refer to the absorption and emission of the UV VIS light by the atoms and various mono atomic ions. It is conceptually similar to the absorption and emission of UV VIS light by molecules. 

Emission spectra for atoms and mono atomic ions are due to gas phase atoms or mono atomic ions in their excited states and these radiates UV VIS lights as they have to release the extra energy while they are returning to their ground state.

Emission spectra are also characterised by very narrow wavelength bands as these are too involved in electronic energy levels in which they get vibrational levels. For a given kind of atom, the emission lines occur at the same wavelengths as the absorption lines because they involve the same energy levels. 

Beer Lambert Law UV VIS Spectrophotometer

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The electromagnetic spectrum has enough energy to promote electronic transition in organic molecules like absorption of UV light (200 to 400 nm). 
The electronic energy levels difference is found to be more than the any other molecular energy levels difference. Therefore, they require radiation of higher energy and short wave length. The relationship between E and λ is a reciprocal one and that short wavelength radiation relates to high energy and long wavelength for lower energy.

Most of the molecules are found in their lowest vibrational states and also of lowest electronic energy level. The UV absorption or the visible light leads to promotion of an electron from E1 to excited electronic level E2. The electronic transition always is given out by both vibrational and rotational form of transition. This helps in promoting electron from ground state to energy level E1. And then further to any vibrational or rotational energy levels of E2.  

Electronic transition are allowed if the orientation of the electron spin does not change during the transition and if symmetry of initial and final are having varying function. These are called the spin and symmetry selection rules. According to Beer Lambert law, the absorbance A of a solution is directly proportional to path length (l) and concentration of absorbing molecule (c) in moles per litre.

A = ε c l 

Where, ε = molar absorptivity of the absorbing molecule, and in some cases it is called the molar extinction coefficient and a characteristic of the molecule. The mathematical physical basis of light absorption measurements on gases and solutions in UV VIS in IR region is also given by same relation. 

A = ε c l = Ig (100 / T %) Ṽ

An absorption spectrum is characterised by two parameters, the maximum position (λ max) and molar extinction coefficient calculated in generally at λ max. The relationship between ε, sample concentration (c) and thickness of absorbing medium is shown by Beer Lambert law.

How Does UV VIS Spectroscopy Work?

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An absorption spectrum is basically a combination of electronic, vibrational and rotational transition and peak spectrum gives out the electronic transition line while the rest is formed by series of lines related to rotational and vibrational forms of transition. Absorption spectra is also sensitive to changes in temperature. With the increase in temperature, the states of rotational as well as vibrational forms for molecules induce widening of spectrum which is recorded. 

A spectrum which is shown from its peak position or the maximum position is given out as: 

δ v = v2 – v1 

The v1 and v2 are the frequency which are equal to half of the maximum intensity. The molecules which fail to display any type of motion, the spectral distribution shows the Lorenz pattern of profile. Thermal motion which are induced gives out different speed displacements and motion for molecules and thus can have different transition probabilities.  

Organic compounds with chromophore contained system absorb in the UV VIS levels. It is usually assumed that the spectroscopic data are known and the determination of single substances is found to be possible by only Beer Lambert law. 

One of the organic components of a mixture often has a relatively low extinction coefficient which does not allow accurate determination. These works out for saturated ketones, aldehydes, carboxylic acids and their respective derivatives which also absorb in analytical forms which are found to be unfavourable spectra below 200 nm. 
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