INTRODUCTION:

Ultraviolet (UV) spectroscopy is a physical technique of the optical spectroscopy that uses light in the visible, ultraviolet, and near infrared ranges, the molecular absorption is studied in the wavelength region of 190 to 800 nm of Electromagnetic spectrum, Ultraviolet region from 190nm - 400nm and visible region from 400nm - 800 nm¹.

When monochromatic radiation passes through a homogeneous solution in a cell, the intensity of the emitted radiation depends upon the thickness (b) and the concentration (C) of the solution. Io is the intensity of the incident radiation and I is the intensity of the transmitted radiation².

Figure 1: Mechanism of absorbance

The amount of radiation absorbed may be measured in a number of ways:

Transmittance, T = I/I0

Transmittance %T = 100 X T

Absorbance A = 2 – log10 %T

The equation, A= 2 – log10 %T, it allows you to calculate absorbance from percentage transmitted data.³

The relationship between absorbance and transmittance is given in following diagram:

So, if all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite⁴.

Principle:

Beer-Lambert Law: The Beer–Lambert law states that the absorbance of a solution (A) is directly proportional to the concentration of the absorbing species(c) in the solution and the path length (b).⁵

Absorbance A = molar absorptivity constant x cell length x concentration

A = abc

C = A /a b

Where,

A = absorbance

A = molar absorptivity

b = path length

c = Concentration

Electronic Transition:

A molecule or ion will exhibit absorption in the visible or ultraviolet region when radiation causes an electronic transition within its structure. Thus, the absorption of light by a sample in the ultraviolet or visible region is accompanied by a change in the electronic state of the molecules in the sample. The energy supplied by the light will promote electrons from their ground state orbitals to higher energy, excited state orbitals or antibonding orbitals⁶.

The following electronic transitions can occur by the absorption of ultraviolet and visible light:

Figure 2: Electronic transistion of σ, π and n electrons

1. σ to σ* Transitions:

An electron in a bonding σ orbital is excited to the corresponding antibonding orbital. The energy required is large. For example, methane (which has only C-H bonds, and can only undergo σ to σ* transitions) shows an absorbance maximum at 125 nm. Absorption maxima due to σ to σ* transitions are not seen in typical UV-Vis. spectra (200 - 700 nm)⁸.

2. n to σ* Transitions:

Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of n to σ* transitions. These transitions usually need less energy than σ to σ*. Transitions They can be initiated by light whose wavelength is in the range 150 - 250 nm.

3. n to π* and π to π* Transitions:

Most absorption spectroscopy of organic compounds is based on transitions of n or π electrons to the π^* excited state. This is because the absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide the π electrons.

Molar absorptivity from π to π* transitions are relatively low, and range from 10 to 100 L mol^-1 cm^-1. π to π* transitions normally give molar absorbtivities between 1000 and 10,000 L mol^-1cm^-1.⁹

Instrumentation:

The Essential components of UV-VIS Spectrophotometer are as follows:

1. Sources (UV and visible)

2. Monochromator

3. Sample containers (Cuvette)

4. Detector

5. Amplifier and recorder

Figure 3: Schematic diagram of UV Spectrophotometer

1. SOURCES:

UV-Vis Spectroscopy requires a continuous source, or one that emits radiation over a broad range of wavelengths. Various UV radiation sources are as follows:

1). Hydrogen lamp:

Hydrogen lamp are stable, robust and emit continuous radiation in range of 160-380 nm.

It Consist of hydrogen gas under high pressure through which there is electrical discharge, hydrogen molecules are excited and emit radiation.

2). Deuterium lamp:

A deuterium lamp is a gas discharge lamp and is often used as a UV source. It emits in radiation in range of 160-450nm.It is more expensive that Hydrogen lamp.

3). Tungsten lamp:

Tungsten Lamp is the most common light source used in spectrophotometer.

It consists of a tungsten filament enclosed in a glass envelope, with a wavelength range of about 330 to 900 nm, are used for the visible region.

4). Xenon discharge lamp:

A xenon lamp is a discharge light source with xenon gas sealed in a bulb. The xenon emits radiation in range of 250-600 nm.

2. MONOCHROMATOR:

A monochromator produces monochromatic light by removing unwanted wavelengths from the radiation source light. Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the entrance slit. The beam is collimated, and then strikes the dispersing element at an angle.

The beam is split into its component wavelengths by the grating or prism. By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit ¹⁰.

Types of monochromator:

1. Prism Monochromator

2. Grating Monochromator.¹¹

Figure 4: Prism monochromator

3. SAMPLE CONTAINERS (CUVETTE):

Cuvettes are sample container which used to hold samples for spectroscopic measurement and which is transparent to all wavelength of light passing through it. The cuvette made of Quartz ,Square shape and having path length 1 cm are selected and can be used for wavelengths ranging from 190 to 200 nm¹³.

4. DETECTORS:

Detector converts light energy into electrical signals that are displayed on readout devices. The transmitted radiation falls on the detector which determines the intensity of radiation absorbed by sample the following types of detectors are employed in instrumentation of absorption spectrophotometer.

Types of Detectors:

1. Barrier layer cell/Photovoltaic cell

2. Phototubes/ Photo emissive tube

3. Photomultiplier tube.¹⁴

General Rule for Performing UV Spectroscopy:

1. Drug Must be completely Soluble in Solvent

2. Drug must absorb UV Visible radiation or light.

3. Drug must not interact with solvent

4. Solvent must be selected on consideration of Cutt off wavelength.

5. The solvent should be UV transparent at the measuring wavelength.

6. When using volatile solvents stoppered cells should be employed to avoid evaporation

leading to changes in solution concentration.

7. Absorbtion must be linear.

8. Only Dilute solution obeys Beer-Lambert law.

9. Calibration curve must be linear.

10. In case of Binary drug, both drug must ne soluble in same solvent.

11. Drug and solvent must be free of contamination.¹⁵

Solvents Cut –off Wavelength:

UV cut off is defined as the wave length where solvent also absorbs light (UV or Visible). In that region, the measurement should be avoided. It is difficult to determine the absorbance comes from your analyte or your solvent.¹⁶

So when choosing a solvent is aware of its absorbance cutoff and where the compound under investigation is thought to absorb. If they are close, chose a different solvent.

The following table provides an example of solvent cut offs.

Table 1: Commonly used solvent and Cut –off Wavelength¹⁷

Solvent	Cut-off (nm)
Is-octane	202
Ethyl alcohol	205
Cyclohexane	200
Acetone	325
Tetrachloroethylene	290
Benzene	280
Carbon tetrachloride	265
Water	180

Application of UV-VIS Spectroscopy¹⁸:

1. Detection of Impurities

2. Structural Illucidation of organic compound

3. Detection of conjugation

4. Detection of functional group

5. Detection of Geomitrical isomer

6. Molucular Weight Determination

7. Distinction of Cis-Trans Isomerism.

REFERENCE:

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Received on 11.01.2020 Modified on 05.02.2020

Research J. Science and Tech. 2020; 12(1): 47-51.

DOI: 10.5958/2349-2988.2020.00005.4