Principle of uv visible spectroscopy

Introduction:

Already we have a basic idea about UV-visible spectroscopy from part-Ⅰ. Now we are going to focus on details about Uv-visible spectroscopy.

The main pillar of UV-visible spectroscopy: Law of Absorption

Here we can see that 100% incident radiation passes through a sample and after that 95% of radiation has been transmitted because 5% of radiation has been absorbed by the sample’s particles.

Case1- If a sample the solution is transparent then we can say,

                                            %A=0%

                                      or, %T=100%

                                          I₀₌I_t [Where, %A=Percentage of absorption, %T= percentage of transmitted radiation, I₀₌ Incident radiation, I_t= Transmitted radiation]

Case2- If a sample the solution is not transparent,

                                            %A= more than 0

                                      or, %T= Less than 100%

                 So here we can say I₀>I_t

Now, A=I₀/I_t

T=I_t/I₀

Then, A= Log₁₀(1/T) ...….... equation (1)

                                           T=I/I₀

1/T=I₀/I………. from equation (1)

                                Then, A=Log₁₀(I₀/I) ………equation (2)

From part 1 of UV visible spectroscopy, we knew from Lambert’s law when a beam of monochromatic radiation passes through an absorbing medium, there is a decrease in the intensity of radiation which is directly proportional to the thickness(pathlength) of the solution.

From Absorption law, we have got, A=Log₁₀(I₀/I)

                                                          A=log₁₀(I₀/I)ꭃl

                                                     or, Aꭃl

                                                     or, A=εl [Where A= Absorption, ε= Molar extinction coefficient or molar absorptivity, l= Thickness (Pathlength)].

And Beer’s law depicts when a beam of monochromatic radiation passes through the absorbing medium, there is a decrease of the intensity of radiation which is directly proportional to the concentration of the solution.

From Absorption law, we have A=Log₁₀(I₀/I)

A=Log₁₀(I₀/I)ꭃC

                                                    or, AꭃC

                                                    or, A=εC [C= Concentration of the sample]

Now Beer-Lambert’s law depicts that when a beam of monochromatic radiation is passed through the absorbing medium, then the decrease in the intensity of radiation is directly proportional to the thickness (Pathlength) and concentration of the solution as well.

From Absorption law, we have gotA=Log₁₀(I₀/I)

                                                   or, A=Log₁₀(I₀/I)ꭃlC

                                                   or, AꭃlC

                                                   or, A=εlC

A=εCl[Where C= Concentration of sample and l=Thickness of the sample].

Principle of UV-Visible spectroscopy:

Here we can see that the highest absorbance reveals at 350 nm and this is the λ_maxof the sample where we can say at which wavelength samples reveal their maximum absorbance it will be the sample’s λ_max.

Electronic transitions in UV-visible spectroscopy:

σ-σ*	n-σ*	π-π*	n-π*
1. Requires energy of 150nm which is very high energy as compared to UV range	1. It goes to the non-bonding(n) to the σ*, so here energy requires approx. 175nm.	1. It starts from π molecular orbital and excites into π*. Always one electron will go from HOMO to LUMO. Energy requires more than 200nm.	1. It starts from n(non-bonding) orbital and it goes to the LUMO of π* orbital. It requires more than 200nm energy.
2. Vacuum UV region (below 200nm) where oxygen starts getting absorbed and pathlength should be free from any air, it is carried out under the vacuum that’s why it is known as vacuum UV region.	2. It occurs when any heteroatom is present in the saturated compounds. e.g. Alcohols, aldehydes, ketones, amines, water, etc.	2. when any double or triple bonded hydrocarbons are present then this transition will occur.	2. It maybe up to 800nm but excitation energy is dependent on the complete conjugation present in that particular compound.
3. All saturated hydrocarbons (methane, propane,etc.) show σ-σ* transition.	3. Water shows absorption at 167nm, Methyl chloride shows absorption at 169nm, Methanol shows absorption at 174nm.	3. Aromatic compounds also show this type of.	3. Carbonyl compounds or any other compounds which are having hetero atom and unsaturation shows this type of transition.

Chromophores, Auxochromes, and Factors affecting absorption:

Chromophores: These are the covalently bonded chemical moieties with any compound liable for the absorption of UV-visible radiations.

e.g. Aldehyde, Ethylene, Carbonyl groups, etc.

Chromophores are categorized into two categories:

1. Chromophores with π-π* transition- e.g. >C=C>, ⸻C≡C⸻C

2. Chromophores with n-π* transition-e.g. ⸻C=O, ⸻N=N⸻

When chromophores are added with any compounds the absorption wavelength will be increased.

Auxochromes: It defines any moiety which doesn’t show any specific color or absorption when separated but when it is attached with any chromophore it increases the absorption wavelength towards the longer wavelength by the formation of the new chromophore.

e.g. OH, NH₂, OR, NHR, ⸻SH, etc.

Suppose, Benzene is a chromophore, but when an NH₂ group is added with it then it will form aniline. Where λ_maxof benzene is 255nm after the addition of this NH₂ group λ_max for aniline is 280nm.

Factors affecting absorption in UV-visible spectroscopy:

1. Absorbing compounds- There are two absorbing compounds, 1- Chromophores and                  2- Auxochromes.
2. Solvent effect- There are various solvents that show absorption in the UV range, e.g. Benzene (255nm), Carbon tetrachloride (265nm), Chloroform (240nm) should be avoided. Whenever we are using UV-visible spectroscopy we have to use those solvents which doesn’t show any absorption in the UV range. So we can use ethanol, methanol, ether, etc.
3. Temperature- Low temperature is suitable for UV-visible spectroscopy which gives higher absorption.
4.  Inorganic moieties: There are two types of inorganic moieties

• Complex inorganic moieties- MnO₄^-
• Single inorganic moieties – Au, Ag, etc.

Absorption and intensity sifts:

We know that seven colors of UV-visible spectroscopy which is VIBGYOR. V (violet) is on the left side known as Blueshift and R (red) is on the right side known as Redshift.

Bathochromic shift: If absorption wavelength is increased towards a redshift or higher wavelength then it is called a bathochromic shift. There are mainly four reasons for bathochromic shifts:

1. Due to the addition of chromophores
2. Auxochrome.
3. Solvent effects- e.g. Carbonyl compound where n-π* transition takes place.
4. Increasing conjugation- Whenever conjugation is increased in any compound, increase in n the double bond or triple bond the wavelength of that particular compound increases towards redshift.

Hypsochromic shift (blue shift): Here compound shifts absorption wavelength towards the shorter wavelength. Four reasons behind this shift, are:

1. Removal of chromophores- If chromophores are removed from any compound then the absorption wavelength of the particular compound will be decreased.
2. Removal of auxochromes.
3. Solvent effects- e.g. Aniline is absorbed in 280nm bin if it dissolves into acid then it is ionized other rm of aniline which is absorbed in 203nm. there is a decrease in 77 nm in the case of aniline.
4. Removal of conjugation- Whenever conjugation from any compound is removed then a particular compound will give hypsochromic shift.

Hyperchromic shift: It means shifting of Molar absorptivity or molar extinction coefficient (ε) towards higher values.

e.g. Pyridine it gives absorption at 257nm, it's εmax=2750. When this pyridine converts into 2-methyl pyridine (attachment of one CH₃ group) due to this CH₃ group absorption wavelength will be 262nm because there will be an increment of 5nm and theεmax= 3660. So this pyridine shows a hyperchromic shift when the addition of one methyl the group takes place.

Hypochromic shift: There is a shifting of εmax value towards the lower values.

e.g. Biphenyl it gives absorption at 250 nm and εmax =19000, but when the addition of one CH₃ group takes place it will covert into 2-methyl-biphenyl which gives absorption at 237nm and εmax = 10250. So, here molar absorptivity will decrease from 19000 to 10250 that’s why it is known as a hypochromic shift.

Hope the Part-1 and Part-2 of UV-Visible Spectroscopy were helpfuwereor you. In the next blog, we will discuss other instruments, and how exactly they work in detail. Till then follow and subscribe @A-Z pharmascience so that you don’t miss any updates.