UV-visible Spectroscopy

       Spectroscopy is a study where we can observe how properties of a matter interact with various types of radiated energy (mainly electromagnetic radiation) of the electromagnetic spectrum. The properties of matter absorb the energy and create an excited state.

Basic of Electromagnetic Radiation:

Wavelength: Distance between two Crests and two Troughs are called as wavelength.                                                        

Denoted by- λ, Unit for wavelength- Ǻ, µ, nm, mµ, etc.               

                                                                                                                          

Wavenumber: It is the number of waves passes through a space of 1 cm.

• It is reciprocal of the wavelength.
• Denoted by- ʋ (number). ʋ = 1/λ
• The unit of wavenumber- cm^-1.

Frequency: It is the number of waves that can pass through a point in a duration of 1sec.

Ø  It is denoted by ʋ and inversely proportional to wavelength.

Ø  ʋ∞1/λ, ʋ=c/λ, c= velocity of EMR(2.998×10¹⁰cm.sec^-1).

Ø  Unit- cycles per sec/sec^-1/hz.

                                           E=hʋ

                                           E=h c/λ, [ʋ=c/λ]

                                           h=Plank’s constant (6.626×10^-27erg.sec).

Basic of UV-Visible Spectroscopy: 

      The term spectroscopy has been coming from Isaac Newton’s optics experiments (1666-1672). Newton used this term “spectrum” to relate the rainbow of colors that assemble to form light and that are transpired when the white light is passed through a prism. At 1800s, Joseph von prepared an experimental advances divergent spectrometer that enabled spectroscopy to be a more appropriate and quantitative scientific technique. Theory of spectroscopy based on two different following states:

Ground state: Where electrons are lying with lowest energy levels, that means the total energy of electrons cannot be lowered by transferring one or more electrons to different orbitals, whereas in Excited state electrons are temporarily occupies energy which is greater than ground state. It becomes excited due to absorption of photons or any kind of energies with a nearby atoms or particle.

Basically, it follows Absorption Law, that means a monochromatic radiation passes through a chamber where contained test sample or solution and the particles of the solution will absorb radiation after that the rest of the amount of radiation will be transit. After all the detector will execute how much light was reflected and transmitted from sample.

Principle of UV-Visible Spectroscopy based on these following laws:

Beer’s Lambert’s law:

Beer’s law: When a beam of monochromatic radiation passed through the absorbing medium, then the decrease of intensity of radiation will be directly proportional to the concentration of solution.

                                          A∞C

                                      A=εC [Where A =Absorption, ε=Molar extinction coefficient (unit=L/mol^-cm) or molar absorptivity, C=Concentration of sample].

Lambert’s Law: When a beam of monochromatic radiation is passed through the absorbing medium, then decrease in the intensity of radiation will be directly proportional to the thickness(pathlength) of the solution.

                                                   A∞l

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

So, Beer’s 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.

                                     A=εCl [Where A =Absorption, ε=Molar extinction coefficient (unit=L/mol^-cm) or molar absorptivity, C=Concentration of sample, l= Pathlength].

INSTRUMENTATION: 

Source: There are two types of Radiation source

                               Single Beam

                               Double Beam

Single beam: It has only single beam of light.

Double beam: Radiation Source- It radiates through the monochromator (converting polychromatic light into monochromatic light).

Two types of lamp are used:

                                          1)   H2 (hydrogen) – D2 (Deuterium) lamp(200-375nm)

                                          2)   Tungsten- Filament Lamp (375nm-800nm)

Monochromator:

• Entrance slit – Generally its cylindrical shaped and internally constructed with rectangular shape used to stop inner reflection of radiation at the moment of entering the lens.
• Lens- It acts on radiation to converge and expand it in singular dimension.
• Gratings/Prism- Converting polychromatic radiation into their seven several parts of color (VIBGYOR).
• Exit slit- Single color radiation passes through this portion to avoid the inner reflection.Chopper- Through the chopper the single radiation is divided into two separate directions.

Cuvette: Cuvette is sample holder which is made up of Quartz (UV-Visible).       

Detectors: The detectors are converts radiation into a proportional electrical signal and quantifying the radiation was transmitted through the sample.

There are three types of detectors have been used:

                                     1) Photomultiplier tube (PMT)

                                     2) Photodiode array (PDA)

                                     3) Charged Coupled Detector (CCD)

1) Photomultiplier tube: Used to detect light of very weak signals which is a photo emissive device and absorption of a photon results in the emission of an electron. Its work by multiplication of electrons through dynode (intermediating electrode where additional electrons are emits in photomultiplier and amplifying device).

2) Photodiode array: Presence of diodes converts the electric signal from radiation at the range of 200nm to 800 nm. Mainly its used in HPLC.

3) Charged Coupled Detector: Photoactive portion of CCD is a layer of polysilicon where silicon surface is used to catch photons. This device converts radiations into digital signals.

Why we need UV-Visible Spectroscopy:

• Quantitative determination of different analytes (e.g.- transitional metal ions, highly conjugated organic compounds, biological macromolecules).
• To detect the impurities, present in the sample solution.
• To elucidate structures of organic compounds.
• To detect the presence of functionals group in the compound.
• To determine the many drugs are either in the form of raw material or formulation.
• To observe the molecular weight of the compounds which can be measured spectrophotometrically by making the suitable derivatives of these compounds.

This is one of the instruments that is used in analytical pharmacy, stay connected to know more.
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